Chimeric infectious dna clones, chimeric porcine circoviruses and uses thereof

ABSTRACT

The present invention relates to infectious DNA clones, infectious chimeric DNA clones of porcine circovirus (PCV), vaccines and means of protecting pigs against viral infection or postweaning multisystemic wasting syndrome (PMWS) caused by PCV2. The new chimeric infectious DNA clone and its derived, avirulent chimeric virus are constructed from the nonpathogenic PCV1 in which the immunogenic ORF gene of the pathogenic PCV2 replaces a gene of the nonpathogenic PCV1, preferably in the same position. The chimeric virus advantageously retains the nonpathogenic phenotype of PCV1 but elicits specific immune responses against the pathogenic PCV2. The invention further embraces the immunogenic polypeptide expression products. In addition, the invention encompasses two mutations in the PCV2 immunogenic capsid gene and protein, and the introduction of the ORF2 mutations in the chimeric clones.

CROSS-REFERENCE TO RELATED U.S. APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 14/940,799, filed on Nov. 13, 2015, allowed, which is a continuationof U.S. application Ser. No. 13/268,008, filed on Oct. 7, 2011, now U.S.Pat. No. 9,211,324, which is a continuation of U.S. application Ser. No.12/539,540, filed on Aug. 11, 2009, now U.S. Pat. No. 8,058,048, whichis a division of U.S. application Ser. No. 11/893,182, filed on Aug. 15,2007, now U.S. Pat. No. 7,575,752, which is a division of U.S.application Ser. No. 10/808,964, filed on Mar. 25, 2004, now U.S. Pat.No. 7,279,166, which, in turn, is a continuation-in-part applicationwhich claims the benefit under 35 U.S.C. § 120 of the prior,nonprovisional U.S. application Ser. No. 10/314,512, filed on Dec. 9,2002, now U.S. Pat. No. 7,276,353, which claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Application No. 60/424,840, filed onNov. 8, 2002, abandoned, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/340,775, filed on Dec. 12,2001, abandoned. All prior applications are incorporated herein byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This study was supported in part by a grant from the U.S. Department ofAgriculture National Research Initiative Competitive Grant Program (NRI2004-35204-14213).

REFERENCE TO A “Sequence Listing”

The material on a single compact disc containing a Sequence Listing fileprovided in the prior nonprovisional application is incorporated byreference. The date of creation is Jan. 22, 2003 and the size isapproximately 9.5 kb.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention concerns infectious porcine circovirus type-1(PCV1) and type-2 (PCV2) DNA clones, chimeric PCV1-2 infectious DNAclones and live chimeric viruses derived from the chimeric DNA clones,useful as vaccines. The invention further concerns two mutations in thePCV2 immunogenic capsid gene and protein, and the introduction of theORF2 mutations in the chimeric clones.

Description of the Related Art

All patents and publications cited in this specification are herebyincorporated by reference in their entirety.

Porcine circovirus (PCV) was originally isolated as a noncytopathic cellculture contaminant of a porcine kidney cell line PK-15 (I. Tischer etal., “A very small porcine virus with circular single-stranded DNA,”Nature 295:64-66 (1982); I. Tischer et al., “Characterization ofpapovavirus and picornavirus-like particles in permanent pig kidney celllines,” Zentralbl. Bakteriol. Hyg. Otg. A. 226(2):153-167 (1974)). PCVis a small icosahedral non-enveloped virus that contains a singlestranded circular DNA genome of about 1.76 kb. PCV is classified in thefamily of Circoviridae along with other animal circoviruses such aschicken anemia virus (CAV), Psittacine beak and feather disease virus(PBFDV) and tentative members columbid circovirus (CoCV) discovered inpigeons, goose circovirus and canary circovirus, and three plantcircoviruses (banana bunchy top virus, coconut foliar decay virus andsubterranean clover stunt virus) (K. V. Phenix et al., “Nucleotidesequence analysis of a novel circovirus of canaries and its relationshipto other members of the genus circovirus of the family Circoviridae,” J.Gen. Virol. 82:2805-2809 (2001); Todd et al., “Genome sequencedeterminations and analyses of novel circoviruses from goose andpigeon,” Virology 286:354-362 (2001); M. R. Bassami et al., “Psittacinebeak and feather disease virus nucleotide sequence analysis and itsrelationship to porcine circovirus, plant circoviruses, and chickenanemia virus,” Virology 249:453-459 (1998); J. Mankertz et al.,“Transcription analysis of porcine circovirus (PCV),” Virus Genes16:267-276 (1998); A. Mankertz et al., “Cloning and sequencing ofcolumbid circovirus (CoCV), a new circovirus from pigeons,” Arch. Virol.145:2469-2479 (2000); B. M. Meehan et al., “Sequence of porcinecircovirus DNA: affinities with plant circoviruses,” J. Gen. Virol.78:221-227 (1997); B. M. Meehan et al., “Characterization of novelcircovirus DNAs associated with wasting syndromes in pigs,” J. Gen.Virol. 79:2171-2179 (1998); D. Todd et al., “Comparison of three animalviruses with circular single-stranded DNA genomes,” Arch. Virol.117:129-135 (1991)).

Members of the three previously recognized animal circoviruses (PCV,CAV, and PBFDV) do not share nucleotide sequence homology or antigenicdeterminants with each other (M. R. Bassami et al., 1998, supra; D. Toddet al., 1991, supra). The genome of the newly identified CoCV sharedabout 40% nucleotide sequence identity with that of PCV (A. Mankertz etal., “Cloning and sequencing of columbid circovirus (CoCV), a newcircovirus from pigeons,” Arch. Virol. 145:2469-2479 (2000)). Recently,a novel human circovirus with a circular genome, designated astransfusion transmitted virus or TT virus (TTV), was identified fromindividuals associated with post-transfusion hepatitis (H. Miyata etal., “Identification of a novel GC-rich 113-nucleotide region tocomplete the circular, single-stranded DNA genome of TT virus, the firsthuman circovirus,” J. Virol. 73:3582-3586 (1999); T. Nishizawa et al.,“A novel DNA virus (TTV) associated with elevated transaminase levels inposttransfusion hepatitis of unknown etiology,” Biochem. Biophys. Res.Commun. 241:92-97 (1997)). Additionally, a human TTV-like mini virus(TLMV) was identified from normal blood donors (P. Biagini et al.,“Genetic analysis of full-length genomes and subgenomic sequences of TTvirus-like mini virus human isolates,” J. Gen. Virol. 82: 379-383(2001); K. Takahashi et al., “Identification of a new human DNA virus(TTV-like mini virus, TLMV) intermediately related to TT virus andchicken anemia virus,” Arch. Virol. 145:979-93 (2000)) and a third novelhuman circovirus, known as SEN virus (SENV), was also discovered fromhumans with post-transfusion hepatitis (T. Umemura et al., “SEN virusinfection and its relationship to transfusion-associated hepatitis,”Hepathology 33:1303-1311 (2001)). The genomic organization of both humanTTV and TLMV is similar to that of the CAV (P. Biagini et al., 2001,supra; H. Miyata et al., 1999, supra; K. Takahashi et al., 2000, supra).Although antibodies to PCV were found in various animal speciesincluding humans, mice, cattle and pigs (G. M. Allan et al.,“Production, preliminary characterization and applications of monoclonalantibodies to porcine circovirus,” Vet. Immunol. Immunopathol.43:357-371 (1994); G. C. Dulac and A. Afshar, “Porcine circovirusantigens in PK-15 cell line (ATCC CCL-33) and evidence of antibodies tocircovirus in Canadian pigs,” Can. J. Vet. Res. 53:431-433 (1989); S.Edwards and J. J. Sands, “Evidence of circovirus infection in Britishpigs,” Vet. Rec. 134:680-1 (1994); J. C. Harding and E. G. Clark,“Recognizing and diagnosing postweaning multisystemic wasting syndrome(PMWS),” Swine Health and Production 5:201-203 (1997); R. K. Hines andP. D. Lukert, “Porcine circovirus: a serological survey of swine in theUnited States,” Swine Health and Production 3:71-73 (1995); G. P. Nayaret al., “Evidence for circovirus in cattle with respiratory disease andfrom aborted bovine fetuses,” Can. Vet. J. 40:277-278 (1999); I. Tischeret al., “Distribution of antibodies to porcine circovirus in swinepopulations of different breeding farms,” Arch. Virol. 140:737-743(1995); I. Tischer et al., “Presence of antibodies reacting with porcinecircovirus in sera of humans, mice, and cattle,” Arch. Virol.140:1427-1439 (1995)), little is known regarding the pathogenesis of PCVin these animal species. Experimental infection of pigs with the PK-15cells-derived PCV did not produce clinical disease and thus, this virusis not considered to be pathogenic to pigs (G. M. Allan et al.,“Pathogenesis of porcine circovirus; experimental infections ofcolostrum deprived piglets and examination of pig foetal material,” Vet.Microbiol. 44:49-64 (1995); I. Tischer et al., “Studies on epidemiologyand pathogenicity of porcine circovirus,” Arch. Virol. 91:271-276(1986)). The nonpathogenic PCV derived from the contaminated PK-15 cellline was designated as porcine circovirus type 1 or PCV1.

Postweaning multisystemic wasting syndrome (PMWS), first described in1991 (J. C. Harding and E. G. Clark, 1997, supra), is a complex diseaseof weaning piglets that is becoming increasingly more widespread. Withthe threat of a potential serious economic impact upon the swineindustry, it has become urgent to develop a vaccine against PCV2, theprimary causative agent of PMWS. PMWS mainly affects pigs between 5-18weeks of age. Clinical PMWS signs include progressive weight loss,dyspnea, tachypnea, anemia, diarrhea, and jaundice. Mortality rate mayvary from 1% to 2%, and up to 40% in some complicated cases in the U.K.(M. Muirhead, “Sources of information on PMWS/PDNS,” Vet. Rec. 150:456(2002)). Microscopic lesions characteristic of PMWS includegranulomatous interstitial pneumonia, lymphadenopathy, hepatitis, andnephritis (G. M. Allan and J. A. Ellis, “Porcine circoviruses: areview,” J. Vet. Diagn. Invest. 12:3-14 (2000); J. C. Harding and E. G.Clark, 1997, supra). PMWS has now been recognized in pigs in Canada, theUnited States (G. M. Allan et al., “Novel porcine circoviruses from pigswith wasting disease syndromes,” Vet. Rec. 142:467-468 (1998); G. M.Allan et al., “Isolation of porcine circovirus-like viruses from pigswith a wasting disease in the USA and Europe,” J. Vet. Diagn. Invest.10:3-10 (1998); G. M. Allan and J. A. Ellis, 2000, supra; J. Ellis etal., “Isolation of circovirus from lesions of pigs with postweaningmultisystemic wasting syndrome,” Can. Vet. J. 39:44-51 (1998); A. L.Hamel et al., “Nucleotide sequence of porcine circovirus associated withpostweaning multisystemic wasting syndrome in pigs,” J. Virol.72:5262-5267 (1998); M. Kiupel et al., “Circovirus-like viral associateddisease in weaned pigs in Indiana,” Vet. Pathol. 35:303-307 (1998); R.Larochelle et al., “Identification and incidence of porcine circovirusin routine field cases in Quebec as determined by PCR,” Vet. Rec.145:140-142 (1999); B. M. Meehan et al., 1998, supra; I. Morozov et al.,“Detection of a novel strain of porcine circovirus in pigs withpostweaning multisystemic wasting syndrome,” J. Clin. Microbiol.36:2535-2541 (1998)), most European countries (G. M. Allan et al.,“Isolation of porcine circovirus-like viruses from pigs with a wastingdisease in the USA and Europe,” J. Vet. Diagn. Invest. 10:3-10 (1998);G. M. Allan and J. A. Ellis, 2000, supra; S. Edwards and J. J. Sands,1994, supra; S. Kennedy et al., “Porcine circovirus infection inNorthern Ireland,” Vet. Rec. 142:495-496 (1998); A. Mankertz et al.,“Characterization of PCV-2 isolates from Spain, Germany and France,”Virus Res. 66:65-77 (2000); C. Rosell et al., “Identification of porcinecircovirus in tissues of pigs with porcine dermatitis and nephropathysyndrome,” Vet. Rec. 146:40-43 (2000); P. Spillane et al., “Porcinecircovirus infection in the Republic of Ireland,” Vet. Rec. 143:511-512(1998); G. J. Wellenberg et al., “Isolation and characterization ofporcine circovirus type 2 from pigs showing signs of post-weaningmultisystemic wasting syndrome in the Netherlands,” Vet. Quart.22:167-72 (2000)) and some countries in Asia (C. Choi et al., “Porcinepostweaning multisystemic wasting syndrome in Korean pig: detection ofporcine circovirus 2 infection by immunohistochemistry and polymerasechain reaction,” J. Vet. Diagn. Invest. 12:151-153 (2000); A. Onuki etal., “Detection of porcine circovirus from lesions of a pig with wastingdisease in Japan,” J. Vet. Med. Sci. 61:1119-1123 (1999)). PMWSpotentially has a serious economic impact on the swine industryworldwide.

The primary causative agent of PMWS is a pathogenic strain of PCVdesignated as porcine circovirus type 2 or PCV2 (G. M. Allan et al.,“Novel porcine circoviruses from pigs with wasting disease syndromes,”Vet. Rec. 142:467-468 (1998); G. M. Allan et al., “Isolation of porcinecircovirus-like viruses from pigs with a wasting disease in the USA andEurope,” J. Vet. Diagn. Invest. 10:3-10 (1998); G. M. Allan et al.,“Isolation and characterisation of circoviruses from pigs with wastingsyndromes in Spain, Denmark and Northern Ireland,” Vet. Microbiol.66:115-23 (1999); G. M. Allan and J. A. Ellis, 2000, supra; J. Ellis etal., 1998, supra; A. L. Hamel et al., 1998, supra; B. M. Meehan et al.,1998, supra; I. Morozov et al., 1998, supra). The complete genomicsequence of the PMWS-associated PCV2 and nonpathogenic PCV1 have beendetermined (R. Larochelle et al., “Genetic characterization andphylogenetic analysis of porcine circovirus type 2 (PCV2) strains fromcases presenting various clinical conditions,” Virus Res. 90:101-112(2002); M. Fenaux et al., “Genetic characterization of type 2 porcinecircovirus (PCV-2) from pigs with postweaning multisystemic wastingsyndrome in different geographic regions of North America anddevelopment of a differential PCR-restriction fragment lengthpolymorphism assay to detect and differentiate between infections withPCV-1 and PCV-2,” J. Clin. Microbiol. 38:2494-503 (2000); A. L. Hamel etal., 1998, supra; J. Mankertz et al., 1998, supra; B. M. Meehan et al.,1997, supra; B. M. Meehan et al., 1998, supra; I. Morozov et al., 1998,supra).

PCV1 is ubiquitous in pigs but is not pathogenic to pigs. In contrast,the genetically related PCV2 is pathogenic and causes PMWS in pigs.Sequence analyses reveals that the PMWS-associated PCV2 typically sharesonly about 75% nucleotide sequence identity with the nonpathogenic PCV1.Some other strains may vary somewhat to about 74% to about 76%nucleotide sequence identity. Both PCV1 and PCV2 have a very similargenomic organization and are small, non-enveloped viruses with a singlestranded circular DNA genome of about 1.76 kb. The PCV genome containsat least two functional open reading frames (ORFs): ORF1 (930 bp)encodes the Rep proteins involved in viral replication (A. K. Cheung,‘Transcriptional analysis of porcine circovirus,” Virology 305: 168-180(2003)) and ORF2 (699 bp) encodes the major immunogenic viral capsidprotein (A. K. Cheung, 2003, supra; P. Nawagitgul et al., “Modifiedindirect porcine circovirus (PCV) type 2-based and recombinant capsidprotein (ORF2)-based ELISA for the detection of antibodies to PCV,”Immunol. Clin. Diagn. Lab Immunol. 9(1):33-40 (January 2002); P.Nawagitgul et al., “Open reading frame 2 of porcine circovirus type 2encodes a major capsid protein,” J. Gen. Virol. 81:2281-2287 (2000)).

Initial attempts to reproduce clinical PMWS in conventional pigs by PCV2inoculation were unsuccessful (M. Balasch et al., “Experimentalinoculation of conventional pigs with tissue homogenates from pigs withpost-weaning multisystemic wasting syndrome,” J. Comp. Pathol.121:139-148 (1999); M. Fenaux et al., “Cloned Genomic DNA of Type 2Porcine Circovirus (PCV-2) Is Infectious When Injected Directly into theLiver and Lymph Nodes of SPF Pigs: Characterization of Clinical Disease,Virus Distribution, and Pathologic Lesions,” J. Virol. 76:541-551(2002)). Experimental reproduction of clinical PMWS in gnotobiotic pigsand conventional pigs with tissue homogenates from pigs with naturallyoccurring PMWS and with cell culture propagated PCV2 produced mixedresults. Clinical PMWS was reproduced in gnotobiotic (SPF) pigs andcolostrum-deprived and caesarian-derived pigs co-infected with PCV2 andporcine parvovirus (PPV) (G. M. Allan et al., “Experimental reproductionof severe wasting disease by co-infection of pigs with porcinecircovirus and porcine parvovirus,” J. Comp. Pathol. 121:1-11 (1999); S.Krakowka et al., “Viral wasting syndrome of swine: experimentalreproduction of postweaning multisystemic wasting syndrome ingnotobiotic swine by coinfection with porcine circovirus 2 and porcineparvovirus,” Vet. Pathol. 37:254-263 (2000)), and in PCV2-inoculatedgnotobiotic pigs when their immune system was activated by keyholehemocyanin in incomplete Freund's adjuvant (S. Krakowka et al.,“Activation of the immune system is the pivotal event in the productionof wasting disease in pigs infected with porcine circovirus-2 (PCV-2),”Vet. Pathol. 38:31-42 (2001)).

Clinical PMWS was also reproduced in cesarean derived/colostrum deprivedpigs (CD/CD) inoculated with PCV2 alone (P. A. Harms et al.,“Experimental reproduction of severe disease in CD/CD pigs concurrentlyinfected with type 2 porcine circovirus and porcine reproductive andrespiratory syndrome virus,” Vet. Pathol. 38:528-539 (2001)) and inconventional pigs co-infected with PCV2 and either porcine parvovirus(PPV) or porcine reproductive and respiratory syndrome virus (PRRSV) (A.Rivora et al., “Experimental inoculation of conventional pigs withporcine reproductive and respiratory syndrome virus and porcinecircovirus 2,” J. Virol. 76: 3232-3239 (2002)). In cases of thePRRSV/PCV2 co-infection, the PMWS characteristic pathological signs suchas lymphoid depletion, granulomatous inflammation and necrotizinghepatitis are induced by PCV2 and not by PRRSV (P. A. Harms et al.,2001, supra). However, clinical PMWS was not reproduced in gnotobioticpigs infected with PCV2 alone (G. M. Allan et al., “Experimentalinfection of colostrums deprived piglets with porcine circovirus 2(PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV)potentiates PCV2 replication,” Arch. Virol. 145:2421-2429 (2000); G. M.Allan et al., “A sequential study of experimental infection of pigs withporcine circovirus and porcine parvovirus: immunostaining of cryostatsections and virus isolation, J. Vet. Med. 47:81-94 (2000); G. M. Allanet al., “Experimental reproduction of severe wasting disease byco-infection of pigs with porcine circovirus and porcine parvovirus,” J.Comp. Pathol. 121:1-11 (1999); M. Balasch et al., 1999, supra; J. Elliset al., “Reproduction of lesions of postweaning multisystemic wastingsyndrome in gnotobiotic piglets,” J. Vet. Diagn. Invest. 11:3-14 (1999);S. Kennedy et al., “Reproduction of lesions of postweaning multisystemicwasting syndrome by infection of conventional pigs with porcinecircovirus type 2 alone or in combination with porcine parvovirus” J.Comp. Pathol. 122:9-24 (2000); S. Krakowka et al., 2001, supra; S.Krakowka et al., 2000, supra; R. M. Pogranichnyy et al.,“Characterization of immune response of young pigs to porcine circovirustype 2 infection,” Viral. Immunol. 13:143-153 (2000)). The virus inoculaused in these studies were either homogenates of tissues from pigs withnaturally occurring PMWS, or virus propagated in PK-15 cell cultures (G.M. Allan et al., “Experimental infection of colostrums deprived pigletswith porcine circovirus 2 (PCV2) and porcine reproductive andrespiratory syndrome virus (PRRSV) potentiates PCV2 replication,” Arch.Virol. 145:2421-2429 (2000); G. M. Allan et al., “A sequential study ofexperimental infection of pigs with porcine circovirus and porcineparvovirus: immunostaining of cryostat sections and virus isolation, J.Vet. Med. 47:81-94 (2000); G. M. Allan et al., “Experimentalreproduction of severe wasting disease by co-infection of pigs withporcine circovirus and porcine parvovirus,” J. Comp. Pathol. 121:1-11(1999); M. Balasch et al., 1999, supra; J. Ellis et al., 1999, supra; S.Kennedy et al., 2000, supra; S. Krakowka et al., 2001, supra; S.Krakowka et al., 2000, supra; R. M. Pogranichnyy et al., 2000, supra).Since tissue homogenates may contain other common swine agents such asPPV and porcine reproductive and respiratory syndrome virus (PRRSV) (G.M. Allan et al., “Experimental infection of colostrums deprived pigletswith porcine circovirus 2 (PCV2) and porcine reproductive andrespiratory syndrome virus (PRRSV) potentiates PCV2 replication,” Arch.Virol. 145:2421-2429 (2000); G. M. Allan et al., “Experimentalreproduction of severe wasting disease by co-infection of pigs withporcine circovirus and porcine parvovirus,” J. Comp. Pathol. 121:1-11(1999); G. M. Allan and J. A. Ellis, 2000, supra; J. A. Ellis et al.,“Coinfection by porcine circoviruses and porcine parvovirus in pigs withnaturally acquired postweaning multisystemic wasting syndrome,” J. Vet.Diagn. Invest. 12:21-27 (2000); C. Rosell et al., 2000, supra), andsince the ATCC PK-15 cell line used for PCV2 propagation waspersistently infected with PCV1 (G. C. Dulac and A. Afshar, 1989,supra), the clinical disease and pathological lesions reproduced inthose studies may not be solely attributable to PCV2 infection (G. M.Allan et al., “Experimental infection of colostrums deprived pigletswith porcine circovirus 2 (PCV2) and porcine reproductive andrespiratory syndrome virus (PRRSV) potentiates PCV2 replication,” Arch.Virol. 145:2421-2429 (2000); G. M. Allan et al., “A sequential study ofexperimental infection of pigs with porcine circovirus and porcineparvovirus: immunostaining of cryostat sections and virus isolation, J.Vet. Med. 47:81-94 (2000); G. M. Allan et al., “Experimentalreproduction of severe wasting disease by co-infection of pigs withporcine circovirus and porcine parvovirus,” J. Comp. Pathol. 121:1-11(1999); G. M. Allan and J. A. Ellis, 2000, supra; J. A. Ellis et al.,2000, supra).

Clinical PMWS has also been reproduced in PCV2-inoculated CDCD pigs whenvaccinated with Mycoplasma hyopneumoniae (G. M. Allan et al.,“Immunostimulation, PCV-2 and PMWS,” Vet. Rec. 147:171-172 (2000)). Tworecent field studies by G. M. Allan et al., “Neonatal vaccination forMycoplasma hyopneumoniae and postweaning multisystemic wasting syndrome:a field trial,” Pig J. 48:34-41 (2001), and S. C. Kyriakis et al., “Theeffects of immuno-modulation on the clinical and pathological expressionof postweaning multisystemic wasting syndrome,” J. Comp. Pathol.126:38-46 (2002), tested the effect of immuno-modulation by Mycoplasmahyopneumoniae vaccine on the development of PMWS in endemic herds, andshowed a significant decrease in PMWS cases in unvaccinated groupscompared to the vaccinated animals. However, another recent study usingconventional SPF piglets under controlled laboratory conditions couldnot reproduce such an effect, suggesting that vaccinations with M.hyopneumoniae may potentially influence the development of clinical PMWSbut it is clearly a secondary role to a PCV2 infection. Based on theseand other studies, PCV2 is nevertheless considered to be the primary butnot the exclusive causative agent of PMWS.

The lack of an infectious virus stock of a biologically pure form ofPCV2 has impeded the understanding of PCV2 pathogenesis and theetiological role of PCV2 in PMWS. Vaccinations against PPV and possiblyPRRSV have not consistently been shown to prevent the onset of PMWS inPCV2 infected pigs. Consequently, finding a safe yet potent vaccine thatspecifically targets PMWS has been difficult. There is a definiteart-recognized need in the veterinary field to produce an efficacious,safe vaccine against PCV2 infections and PMWS.

U.S. Pat. No. 6,287,856 (Poet et al.) and WO 99/45956 concern nucleicacids from psittacine beak and feather disease virus (BFDV), acircovirus that infects avian species, and from porcine circovirus(PCV). The patent proposes vaccine compositions comprising naked DNA ormRNA and discloses a nucleic acid vector for the transient expression ofPCV in a eukaryotic cell comprising a cis-acting transcription ortranslation regulatory sequence derived from the human cytomegalovirusimmediate or early gene enhancer or promoter functionally linked to anucleic acid of the sequence. However, since the PCV DNA is derivedsolely from the PK-15 cell line, it is likely to comprise thenonpathogenic PCV1 discovered nearly 30 years ago by I. Tischer et al.,1974, supra, and, therefore, it is not likely to be effective ineliciting an immune reaction to PCV2 or infections caused by PCV2.Subunit vaccines of recombinant proteins made from vectors comprisingopen reading frames are also suggested in the patent but the openreading frames from PCV are not well characterized or distinguished fromeach other. Since the source of the PCV DNA is PK-15 cells, the proteinsmade from those vectors comprising the open reading frames of PCV1 wouldnot possess reliable immunogenic properties, if any, against PCV2.

U.S. Pat. No. 6,217,883 (Allan et al.) and French Patent No. 2,781,159Brelate to the isolation of five PCV strains from pulmonary or ganglionicsamples taken from pigs infected with PMWS in Canada, California andFrance (Brittany), and their use in combination with at least oneporcine parvovirus antigen in immunogenic compositions. Proteins encodedby PCV2 open reading frames (ORF) consisting of ORF1 to ORF13 arebroadly described in the patent but there is no exemplification of anyspecific protein exhibiting immunogenic properties. The patent furtherdiscloses vectors consisting of DNA plasmids, linear DNA molecules andrecombinant viruses that contain and express in vivo a nucleic acidmolecule encoding the PCV antigen. Several other references, forexample, U.S. Pat. No. 6,391,314 B1; U.S. Pat. No. 6,368,601 B1; FrenchPatent No. 2,769,321; French Patent No. 2,769,322; WO 01/96377 A2; WO00/01409; WO 99/18214; WO 00/77216 A2; WO 01/16330 A2; WO 99/29871;etc., also describe the administration of PCV1 or PCV2 polypeptides orthe nucleic acids encoding the polypeptides of various strains.

However, the nonpathogenic PCV1 will not be useful against PCV2infections and the pathogenic PCV2 strains described in the art, even ifattenuated, are likely to be of limited value due to the usual tendencyof a live virus to revert to its virulent state. Therefore, there isstill a long-standing need in the art for a live, infectious,nonpathogenic antigen for the inoculation of pigs against seriousinfection or PMWS caused by PCV2 that is efficacious and remains safe inveterinary vaccines. These goals are met by the construction of the newlive chimeric porcine circovirus described herein, which is based uponthe genomic backbone of the nonpathogenic PCV1 isolated by I. Tischer etal. almost 30 years ago. The novel chimeric porcine circovirus of thepresent invention is able to satisfy that long-standing need by uniquelyand advantageously retaining the nonpathogenic phenotype of PCV1 buteliciting specific immune response against pathogenic PCV2.

PCV2 causes pathological lesions characteristic of PMWS inspecific-pathogen-free (SPF) pigs whereas PCV1 does not (M. Fenaux etal., 2002, supra). Based on the current studies, it is also observedthat cell culture-derived PCV1 replicates more efficiently in PK-15cells than PCV2 (see also M. Fenaux et al., “Immunogenicity andpathogenicity of the chimeric infectious DNA clones between pathogenictype 2 porcine circovirus (PCV2) and non-pathogenic PCV1 in weaningpigs,” J. Virol. 77:11232-11243 (2003)). However, the geneticdeterminants for PCV2 pathogenicity in pigs and for the enhanced growthability of PCV1 in PK-15 cells are not known. Thus, another set ofobjectives of the present invention is to identify and characterize thegenetic determinants for PCV2 pathogenicity in vivo and for replicationin vitro.

BRIEF SUMMARY OF THE INVENTION

The present invention concerns infectious chimeric DNA clones of porcinecircovirus (PCV) and live chimeric viruses derived from the DNA clonesthat are useful as vaccines. The new live chimeric, geneticallyavirulent viruses are made from the nonpathogenic PCV1 genomic structurein which an immunogenic gene of a pathogenic PCV2 strain replaces a geneof the PCV1, typically in the same corresponding position. The inventionencompasses the biologically functional plasmids, viral vectors and thelike that contain the new recombinant nucleic acid molecules describedherein, suitable host cells transfected by the vectors comprising theDNA and the immunogenic polypeptide expression products. Included withinthe scope of the present invention is a novel method of protecting pigsagainst viral infection or postweaning multisystemic wasting syndrome(PMWS) caused by PCV2 comprising administering to a pig in need of suchprotection an immunologically effective amount of a vaccine comprising,for example, the cloned chimeric DNA in a plasmid, a chimeric virusderived from the chimeric DNA clone, the polypeptide products expressedfrom the DNA described herein, etc. A further embodiment of theinvention is drawn to novel mutants of the PCV2 immunogenic capsid geneand protein, and the introduction of the mutations in the chimericclones to facilitate cell culture growth and ensure vaccine safety. Theinvention also provides new infectious PCV2 molecular DNA and reciprocalchimeric DNA clones of PCV that find use as experimental models inobtaining or characterizing the novel avirulent viral vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

The background of the invention and its departure from the art will befurther described hereinbelow with reference to the accompanyingdrawings, wherein:

FIG. 1 represents the construction of an infectious PCV2 molecular DNAclone. The relative positions of the primer pair used to amplify thecomplete PCV2 genome are indicated by the arrows (reverse primerPCVSAC2, forward primer PCVSAC2). The PCV2 genomic DNA amplified by PCRis digested with SacII restriction enzyme, and purified. The purifiedand SacII-digested genomic DNA is ligated to form concatemers. Ligatedconcatemers are separated by gel electrophoresis, the tandem genomedimer of PCV2 is purified and cloned into pSK vector that ispre-digested with SacII enzyme to produce a molecular PCV2 DNA clone.

FIGS. 2A and 2B illustrate that the cloned PCV2 plasmid DNA isinfectious when transfected in vitro in PK-15 cells. FIG. 2A shows thedetection of PCV2 antigen by immunofluorescence assay (IFA) in PK-15cells transfected with the cloned PCV2 plasmid DNA. Intenseimmunolabeling of PCV2 antigen is visualized in the nucleus, and to alesser degree, cytoplasm of the transfected cells. FIG. 2B showsmock-transfected PK-15 cells.

FIG. 3A shows the lungs from a pig inoculated by intralymphoid routewith PCV2 DNA at 21 DPI. The lungs are rubbery, failed to collapse, andare mottled tan-red. Tracheobronchial lymph nodes are markedly enlargedand tan (arrows). FIG. 3B represents a microscopic section of a normallung from a control pig (25X). FIG. 3C represents a microscopic sectionof the lung from the pig in FIG. 3A. Note the peribronchiolarlymphohistiocytic inflammation and mild necrotizing bronchiolitis (25X).FIG. 3D illustrates the immunohistochemical staining of the lung in FIG.3A. Note the PCV2 antigen in macrophages (arrows) and fibroblast-likecells (arrow heads) around airways (64X).

FIG. 4A shows a normal lymph node from a control pig. Note thewell-defined lymphoid follicles (arrows) (25X). FIG. 4B represents amicroscopic section of the tracheobronchial lymph node from the pig inFIG. 3A inoculated 21 days previously by intralymphoid route with clonedPCV2 genomic DNA. Lymphoid follicles are poorly defined, there ismild-to-moderate lymphoid depletion, and mild multifocal granulomatousinflammation (25X). FIG. 4C represents a microscopic section of thelymph node in FIG. 4B in a larger magnification focusing on onefollicle. Note the poorly defined follicle with macrophages and giantcells (arrow) replacing follicular lymphocytes (64X). FIG. 4Dillustrates the immunohistochemical detection of PCV2 antigen in thesame lymph node as FIG. 4B in macrophages (arrows) and giant cells(small arrowheads), and dendritic-like cells (large arrowheads) in thefollicles (64X).

FIG. 5 illustrates the construction of a chimeric PCV1-2 (PCV1/PCV2) DNAclone with the nonpathogenic PCV1 genome carrying the immunogenic ORF2capsid gene of the pathogenic PCV2. The dimmerized DNA clone is used forin vitro transfection of PK-15 cells to produce live chimeric virusexpressing ORF2 protein of PCV2, and in vivo animal experiments toconfirm activity.

FIG. 6 represents the construction and organization of the infectiousPCV1, PCV2, chimeric PCV1-2 and reciprocal chimeric PCV2-1 molecular DNAclones. The PCV2 DNA clone is constructed by ligating two full-lengthlinear PCV2 genomes in tandem into the Bluescript SK vector (pSK) by thegeneral methods described previously (M. Fenaux et al., 2002, supra).PCV1 DNA clone is constructed by ligating two full-length linear PCV1genomes in tandem into pSK vector. Chimeric PCV1-2 DNA clone isconstructed by replacing the ORF2 capsid gene of PCV1 with that of thePCV2 in the nonpathogenic PCV1 genomic backbone in pSK vector.Reciprocal chimeric PCV2-1 DNA clone is constructed by replacing theORF2 capsid gene of the pathogenic PCV2 with that of the nonpathogenicPCV1 in the pathogenic PCV2 genomic backbone in pSK vector. Bothchimeric clones are dimmers in pSK vector. The arrows represent therelative locations of the PCR primers for the detection of PCV1, PCV2,PCV1-2 and PCV2-1 viremia in inoculated animals.

FIGS. 7A-7J demonstrate that the PCV1, PCV2, chimeric PCV1-2 andreciprocal chimeric PCV2-1 DNA clones are infectious and expressrespective viral antigens when transfected in vitro in PK-15 cells. Theleft panel (7A, 7C, 7E, 7G and 7I) is stained with monoclonal antibodyagainst the PCV1 ORF2. The right panel (7B, 7D, 7F, 7H and 7J) isstained with antibody against PCV2. Panels 7A and 7B are mocktransfected PK-15 cells. Panels 7C and 7D are PK-15 cells transfectedwith the PCV1 DNA clone. Panels 7E and 7F are PK-15 cells transfectedwith the PCV2 DNA clone. Panels 7G and 7H are PK-15 cells transfectedwith the chimeric PCV1-2 DNA clone. Panels 7I and 7J are PK-15 cellstransfected with the reciprocal chimeric PCV2-1 DNA clone.

FIG. 8 represents the full-length DNA sequence of the cloned PCV2molecular DNA (which corresponds to SEQ ID NO:1).

FIG. 9 represents the full-length DNA sequence of the cloned chimericPCV1-2 DNA (which corresponds to SEQ ID NO:2).

FIG. 10 represents the 702 bp (699 bp sequence plus the 3 nucleotidestop codon) DNA sequence of the immunogenic ORF2 capsid gene of thecloned chimeric PCV1-2 DNA (which corresponds to SEQ ID NO:3).

FIG. 11 represents the putative amino acid translation of theimmunogenic ORF2 capsid gene of the chimeric PCV1-2 DNA (whichcorresponds to SEQ ID NO:4).

FIG. 12 shows one-step growth curves of PCV1, PCV2 VP1 and PCV2 VP120.Duplicate synchronized PK-15 cell cultures are each infected with PCV1,PCV2 VP1 or PCV2 VP120, all at an M.O.I. of 0.1. All three viruses havea titer of about 10^(1.5) TCID₅₀/ml at 12 hours postinoculation. PCV1and PCV2 VP120 replicate more efficiently in vitro than PCV2 VP1 does(p=0.0053).

FIG. 13 is a schematic diagram of amino acid mutations in the capsidprotein during serial passages of PCV2 in PK-15 cells. Serial passagenumbers are indicated as VP1, VP30, VP60, VP90 and VP120. Known fieldisolates of PCV2 and PCV1 from different geographic origins are alsocompared for these two mutations.

FIG. 14 shows the quantitative real-time PCR results of PCV2 VP1 andPCV2 VP120 viral genomic copy loads in 1 ml of serum sample collected at−1, 7, 14, 21, 28, 35 and 42 days post inoculation (DPI) from Groups 1,2 and 3 pigs. Group 2 pigs inoculated with PCV2 VP120 that are positivefor PCV2 DNA are indicated with a σ. The numbers (10, 9, 8, 6, 5, 5)inside the symbol Δ on the X-axis indicate the number of pigs in Group 2that are negative for PCV2 viremia on the respective DPI. Group 3 pigsinoculated with PCV2 VP1 that are positive for PCV2 DNA are indicatedwith symbol O. The numbers (3, 2, 2, 2, 2, 4) inside the symbol O on theX-axis indicate the number of pigs in Group 3 that are negative for PCV2viremia on the respective DPI. The PCV2 genomic copy loads arerepresented as a log of copy numbers per 1 ml of serum (Y-axis).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided infectiousmolecular and chimeric nucleic acid molecules of porcine circovirus(PCV), live chimeric viruses produced from the chimeric nucleic acidmolecule and veterinary vaccines to protect pigs from viral infection orpostweaning multisystemic wasting syndrome (PMWS) caused by PCV2. Theinvention further provides immunogenic polypeptide expression productsthat may be used as vaccines. Also described are two novel nucleotidemutations of the PCV2 immunogenic capsid gene resulting in two novelamino acid mutations of the PCV2 immunogenic capsid protein that areshown to be responsible for increased growth rate in vitro andattenuation of virulence in vivo, and therefore find beneficial use inthe chimeric nucleic acid molecule of this invention.

The new avirulent, infectious chimeric DNA molecule of PCV (PCV1-2)comprises a nucleic acid molecule encoding an infectious, nonpathogenicPCV1 that contains an immunogenic open reading frame (ORF) gene of apathogenic PCV2 in place of an ORF gene in the PCV1 genome. Theinfectious chimeric PCV1-2 DNA clone preferably contains the immunogeniccapsid gene (ORF2) of the PCV2 DNA cloned in the genomic backbone of theinfectious, nonpathogenic PCV1 DNA clone. Generally, the capsid gene ofthe PCV2 DNA replaces the ORF2 gene of the PCV1 DNA in the nonpathogenicPCV1 genomic structure but it is contemplated that a variety ofpositional permutations may be constructed through genetic engineeringto obtain other avirulent or attenuated chimeric DNA clones. Thereciprocal chimeric infectious PCV2-1 DNA clone between PCV1 and PCV2 isdisclosed as a control to analyze the chimeric PCV1-2 clone of theinvention and is constructed by replacing the capsid gene of PCV2 withthat of PCV1 in the backbone of the pathogenic PCV2 infectious DNAclone. In addition to being an experimental model, the reciprocalchimeric PCV2-1 DNA clone may find use in making specially tailoredvaccines.

The cloned genomic DNA of PCV2 described herein is shown to be in vitroand in vivo infectious when transfected into PK-15 cells and given topigs. The infectious PCV2 DNA clone produces pathological lesionscharacteristic of PMWS in pigs allowing for an improved characterizationof clinical disease and understanding of virus distribution in thetissue cells. This new, readily reproducible pathogenic agent lendsitself to the development of a suitable vaccination program to preventPMWS in pigs.

The novel chimeric PCV1-2 DNA clone is also infectious by both in vitrotransfection of PK-15 cells and in vivo administration to pigs. Intransfected PK-15 cells, the chimeric PCV1-2 DNA clone expresses thePCV2 capsid antigen (the immunogenic capsid protein of PCV2) whereas thereciprocal chimeric PCV2-1 DNA clone expresses the PCV1 capsid antigen,which is demonstrated by immunofluorescence assay (IFA) using antibodiesspecific to PCV1 or PCV2 capsid antigen. Seroconversion to PCV2-specificantibody is detected in pigs inoculated with the infectious PCV2 cloneas well as the chimeric PCV1-2 clone. Detecting the seroconversion toPCV2-specific antibody establishes that the chimeric PCV1-2 DNA cloneinduces the PCV2-specific antibody in infected pigs and, consequently,acts to protect inoculated pigs from infection with PCV2.

The below examples describe the evaluation of the immunogenicity andpathogenicity of the chimeric DNA clones in inoculated pigs in moredetail. Basically, seroconversions to antibodies against PCV2 ORF2antigen are detected in pigs inoculated with the PCV2 DNA clone (Group3) and the chimeric PCV1-2 DNA clone (Group 4). All of the pigsinoculated with the PCV1 clone and the reciprocal chimeric PCV2-1 DNAclone (Groups 2 and 5, respectively) seroconvert to the PCV1 antibody.The viruses recovered from selected pigs in each group are partiallysequenced and confirmed to be the authentic respective infectious DNAclones used in the inoculation. Gross and microscopic lesions in varioustissues of animals inoculated with the PCV2 DNA clone are significantlymore severe than those found in pigs inoculated with PCV1, chimericPCV1-2 and reciprocal chimeric PCV2-1 DNA clones.

Surprisingly and advantageously, the chimeric PCV1-2 infectious DNAclone having the immunogenic capsid gene (ORF2) of the pathogenic PCV2cloned into the nonpathogenic PCV1 genomic backbone induces a specificantibody response to the pathogenic PCV2 capsid antigen while ituniquely retains the nonpathogenic nature of PCV1 in pigs. Animalsinoculated with the chimeric PCV1-2 infectious DNA clone develop a mildinfection resembling that of PCV1 inoculated animals whileseroconverting to the antibody against the ORF2 capsid protein of thepathogenic PCV2. The average length of viremia observed in PCV1 andchimeric PCV1-2 inoculated animals is shorter, 0.625 weeks and 1 weekrespectively, than that in pathogenic PCV2 inoculated animals which isabout 2.12 weeks. The lack of detectable chimeric PCV1-2 viremia in someinoculated animals does not affect seroconversion to antibody againstPCV2 ORF2 capsid protein in the PCV1-2 inoculated pigs (Group 4). Theresults indicate that, even though the chimeric PCV1-2 viremia is shortor undetectable in some inoculated animals, the chimeric PCV1-2 virus isable to induce antibody response against PCV2 ORF2 capsid protein. Thespecial ability of the chimeric PCV1-2 infectious DNA clone to inducethe immune response specific to the pathogenic PCV2 immunogenic ORF2capsid protein yet remain nonpathogenic to pigs makes the chimericPCV1-2 clone particularly useful as a genetically engineeredlive-attenuated vaccine and other types of vaccines.

The novel, purified and isolated nucleic acid molecules of thisinvention comprise the full-length DNA sequence of the cloned chimericPCV1-2 DNA set forth in SEQ ID NO:2, shown in FIG. 9 and deposited inthe American Type Culture Collection under Patent Deposit DesignationPTA-3912; its complementary strand (i.e., reverse and opposite basepairs) or the nucleotide sequences having at least 95% homology to thechimeric nucleotide sequence (i.e., a significant active portion of thewhole gene). Conventional methods that are well known in the art can beused to make the complementary strands or the nucleotide sequencespossessing high homology, for instance, by the art-recognized standardor high stringency hybridization techniques. The purified and isolatednucleic acid molecule comprising the DNA sequence of the immunogeniccapsid gene of the cloned chimeric PCV1-2 DNA is also set forth in SEQID NO:3 and FIG. 10.

Suitable cells containing the chimeric nucleic acid molecule uniquelyproduce live, infectious chimeric porcine circoviruses. The live,infectious chimeric virus is derived from the chimeric DNA clone bytransfecting PK-15 cells via in vitro and in vivo transfections asillustrated herein. A preferred example of the cloned chimeric PCV1-2DNA is the nucleotide sequence set forth in SEQ ID NO:2 and FIG. 9. Theinvention further envisions that the chimeric virus is derived from thecomplementary strand or the nucleotide sequences having a high homology,at least 95% homology, to the chimeric nucleotide sequence.

Also included within the scope of the present invention are biologicallyfunctional plasmids, viral vectors and the like that contain the newrecombinant nucleic acid molecules described herein, suitable host cellstransfected by the vectors comprising the chimeric and molecular DNAclones and the immunogenic polypeptide expression products. Aparticularly preferred immunogenic protein has the amino acid sequenceset forth in SEQ ID NO:4 and FIG. 11. The biologically active variantsthereof are further encompassed by the invention. One of ordinary skillin the art would know how to modify, substitute, delete, etc., aminoacid(s) from the polypeptide sequence and produce biologically activevariants that retain the same, or substantially the same, activity asthe parent sequence without undue effort.

To produce the immunogenic polypeptide products of this invention, theprocess may include the following steps: growing, under suitablenutrient conditions, prokaryotic or eukaryotic host cells transfectedwith the new recombinant nucleic acid molecules described herein in amanner allowing expression of said polypeptide products, and isolatingthe desired polypeptide products of the expression of said nucleic acidmolecules by standard methods known in the art. It is contemplated thatthe immunogenic proteins may be prepared by other techniques such as,for example, biochemical synthesis and the like.

Another embodiment of the present invention relates to novel mutationsof the nucleotide and amino acid sequences of the PCV2 immunogeniccapsid gene and protein. PCV2 is the primary causative agent ofpostweaning multisystemic wasting syndrome (PMWS), whereas the PK-15cell culture-derived porcine circovirus type 1 (PCV1) is nonpathogenicto pigs. The molecular mechanisms of PCV2 replication and pathogenesishave been poorly understood. As fully described herein, the importantidentification and isolation of two amino acid mutations within the PCV2capsid protein that are vital for PCV2 pathogenicity in vivo andimproved growth ability in PK-15 cells provides a viable mechanism bywhich the chimeric PCV1-2 vaccine of the present invention can growbetter in cell cultures or be made safer in vaccinations.

To identify genetic determinants for virulence and replication, apathogenic PCV2 isolate is serially passaged for 120 times in PK-15cells, and the viruses that are harvested from passages 1 (VP1) and 120(VP120) are biologically, genetically and experimentally characterized.A one-step growth curve is used to compare the growth characteristics ofPCV1, PCV2 VP1, and PCV2 VP120. The results show that the PCV2 VP120virus replicates to a similar titer as PCV1 but more efficiently thanPCV2 VP1 in PK-15 cells with at least 1 log difference. The completegenomic sequences of viruses at passages 0, 30, 60, 90, and 120 aredetermined. Two novel amino acid mutations are identified in the capsidgene after 120 passages, and it is shown that these two mutations areresponsible for the increased growth rate in vitro and the attenuationof virulence in vivo. There are only two nucleotide differences as well,cytosine to guanine (C to G) and adenine to cytosine (A to C), both ofwhich are non-silent mutations that result in the two amino acidchanges. The first mutation occurs at passage 30, in which a proline atposition 110 of the capsid protein is substituted for an alanine(P110A), and this mutation remains in the subsequent passages. Inposition 328 of the nucleotide sequence, cytosine changes to guanine (Cto G) leading to this amino acid change of P110A. The second mutation, asubstitution of an arginine for a serine at position 191 of the capsidprotein (R191S), appears at passage 120 but not in earlier passages. Innucleotide position 573, adenine changes to cytosine (A to C) leading tothis second amino acid change of R191S.

To characterize the pathogenicity of the VP120 virus, 31specific-pathogen-free (SPF) pigs are randomly divided into threegroups. Ten pigs in Group 1 receive phosphate buffered saline asnegative controls. Eleven Group 2 pigs are inoculated intramuscularlyand intranasally with 10^(4.9)TCID₅₀ of PCV2 VP120. Ten pigs in Group 3are inoculated with 10^(4.9)TCID₅₀ of PCV2 VP1. PCV2 viremia is detectedin 9/10 pigs in the PCV2 VP1 group, but only in 4/11 pigs in PCV120group. The viremia in VP1 group (mean 3 weeks) lasts longer than that ofVP120 group (mean 1.6 weeks). In addition, the PCV2 genomic copy loadsin serum, as determined by quantitative real-time PCR, in the PCV2 VP1group are higher than those in PCV2 VP120 group (p<0.0001). Gross andhistopathologic lesions found in pigs inoculated with PCV2 VP1 are moresevere than those inoculated with PCV2 VP120 at both 21 and 42 DPInecropsies (p=0.0032 and p=0.0274, respectively). Taken together, theresults demonstrate that the P110A and R191S mutations in the capsid ofPCV2 enhance the growth ability of PCV2 in vitro and attenuate the virusin vivo.

As a result of the P110A and R191S mutations in the capsid protein, PCV2VP120 replicates more efficiently (p=0.0053) in PK-15 cells with atleast 1 log difference in infectious titer compared to the passage 1virus. PCV2 VP120 replicates to a similar level with the PK-15 cellculture-adapted PCV1, and thus these two mutations either alone orcollectively are responsible for the enhanced growth of PCV2 VP120 invitro. Allan et al. (G. M. Allan et al., 1994, supra) attempted toinfect human Vero cells with PCV1. Intranuclear immune staining,characteristic of PCV1 replication, was not detected until the 6^(th)cell culture passage in Vero cells. By passage 15, PCV1 replicated inVero cells similarly to PK-15 cells. However, it has not been previouslyreported in the literature that 120 passages of the PCV2 in the PK-15cell line will result in significantly improved replication efficiencyto mimic the PK-15 cell culture-adapted PCV1.

Surprisingly, when SPF piglets are inoculated with the PCV2 VP120, fewerpigs develop viremia with shorter duration and lower PCV2 genomic copyloads compared to PCV2 VP1 inoculated pigs. It is shown that thenonpathogenic PK-15 cell-adapted PCV1 has a short average viremia lengthof 0.625 weeks in infected pigs. Analyses of the gross, microscopic andIHC mean scores reveal that the PCV2 VP120 virus inoculated pigs havemilder pathological lesions and clinical signs than the PCV2 VP1 virusinoculated pigs. Taken together, the results from this invention showthat PCV2 VP 120 has advantageously been adapted to grow better in PK-15cells and is attenuated in pathogenicity in vivo.

After 120 passages in PK-15 cells, only two amino acid mutations aredetected in the entire PCV2 genome, suggesting that the PCV2 genome isrelatively stable. This may explain why the sequences of all known PCV2field isolates identified to date are very conserved (M. Fenaux et al.,2002, supra; R. Larochelle et al., 2002, supra). The P110A mutationsurprisingly occurs early (passage 30) during the serial passage, andinvolves two hydrophobic amino acids, proline and alanine. The changefrom proline to alanine may alter the tertiary structure of the capsidprotein as proline is often involved in the bending regions of proteinstructures. This P110A mutation is then retained in the subsequenthigher passages including passage 120. The uniqueness of the retainedP110A mutation through passage 120 compared to the sequences of knownPCV1 and PCV2 field isolates strongly correlates the mutation with abiological role. The R191S mutation occurs very late during the serialpassage (between passages 90 and 120), and is also unique to the VP120as the known PCV2 and PCV1 isolates do not have a serine residue at thisposition. Only glycine, alanine and threonine substitutions at thisposition have been previously identified in field isolates of PCV1 andPCV2.

Amino acid substitutions induced by cell culture passage or chemicalmutagenesis techniques have been routinely used for the attenuation ofmany viruses, and have led to the productions of many vaccines (G. F.Brooks et al., “Pathogenesis & control of viral diseases,” In: Jawetz,Melnick, & Adelberg's Medical Microbiology, 21^(st) Ed. (PublishersAppleton & Lange) 30: 363-365 (1998)). A single amino acid substitutioncan lead to the attenuation of a virus. For example, it has beenpreviously reported that substitution of a proline for a leucine atresidue 101 of the nonstructural 4B protein of the mosquito-borne Dengue4 (DEN4) virus resulted in decreased viral replication in mosquito but aproportionally increased replication in human Vero cells (K. A. Hanleyet al., “A trade-off in replication in mosquito versus mammalian systemsconferred by a point mutation in the NS4B protein of dengue virus type4,” Virology. 312:222-232 (2003)). Hence, the balancing control ofefficient replication of DEN4 virus in either mosquito or human Verocells was maintained by a single amino acid change. In the Circoviridaefamily, a single amino acid mutation in the VP1 capsid protein of CAVwas found to be responsible for the pathogenicity of the virus inchickens (S. Yamaguchi et al., “Identification of a genetic determinantof pathogenicity in chicken anaemia virus,” J. Gen. Virol. 82: 1233-1238(2001)). Taken together, the results from the present invention stronglycorrelate the mutation to biological activity and demonstrate thatP110A, R191S or collectively, is responsible for the attenuation of PCV2VP120 in pigs or for the improved growth ability of PCV2 in PK-15 cells.

Since the results from this invention demonstrate that the P110A andR191S mutations in the capsid of PCV2 enhance the growth ability of PCV2in vitro and attenuate the virus in vivo, the P110A and R191S mutations(both singly and collectively) may be advantageously introduced into thecapsid gene of the chimeric PCV1-2 vaccine to make the chimera PCV1-2vaccine of the invention grow better in cell cultures or make it saferin pigs. This is accomplished by inserting the mutated immunogeniccapsid gene containing the novel P110A and R191S mutations in thechimeric clones in lieu of using the ORF2 of the pathogenic PCV2. Themutations are introduced into the capsid gene of the PCV1-2 vaccineusing art-recognized techniques such as those found in the instructionmanual for the QuikChange® Multi Site-Directed Mutagenesis Kitcommercially available from Stratagene Inc., La Jolla, Calif.Alternatively, the mutated PCV2 ORF2 for use in the chimeric virus maybe made by well-known biochemical synthesis processes to substitute oneor both of the proline and arginine with the alanine and serine aminoacids at positions 110 and 191, respectively, of the immunogenic capsidprotein. The final mutant clones may be readily sequenced to ensure thatthe intended P110A, R191S or both mutations are properly introduced andthere is no other unwanted mutation. The PCV1-2 vaccine containing themutation may be further tested in cell culture by routine procedures toselect the combination that facilitates cell culture growth or ensuresimproved safety measures when vaccinating pigs due to the furtherattenuation of PCV2 virulent properties, if any persist. While thebenefit of the PCV1-2 chimera lies in its natural avirulent trait, thealternative use of the mutated PCV2 ORF2 to make the PCV1-2 chimeraprovides another embodiment of the present invention that is availableif further safening of the natural live chimera vaccine becomeswarranted.

Vaccines of the chimeric viral and molecular DNA clones, and methods ofusing them, are also included within the scope of the present invention.Inoculated pigs are protected from serious viral infection and PMWScaused by PCV2. The novel method protects pigs in need of protectionagainst viral infection or PMWS by administering to the pig animmunologically effective amount of a vaccine according to theinvention, such as, for example, a vaccine comprising an immunogenicamount of the chimeric PCV1-2 DNA, the cloned chimeric virus, a plasmidor viral vector containing the chimeric DNA of PCV1-2, the polypeptideexpression products, the recombinant PCV2 DNA, etc. Other antigens suchas PRRSV, PPV, other infectious swine agents and immune stimulants maybe given concurrently to the pig to provide a broad spectrum ofprotection against viral infections.

The vaccines comprise, for example, the infectious chimeric PCV1-2 DNA,the cloned PCV chimeric DNA genome in suitable plasmids or vectors suchas, for example, the pSK vector, an avirulent, live chimeric virus, aninactivated chimeric virus, etc. in combination with a nontoxic,physiologically acceptable carrier and, optionally, one or moreadjuvants. The vaccine may also comprise the infectious PCV2 molecularDNA clone described herein. The infectious chimeric PCV1-2 DNA, theplasmid DNA containing the infectious chimeric viral genome and the livechimeric virus are preferred with the live chimeric virus being mostpreferred. The avirulent, live viral vaccine of the present inventionprovides an advantage over traditional viral vaccines that use eitherattenuated, live viruses which run the risk of reverting back to thevirulent state or killed cell culture propagated whole virus which maynot induce sufficient antibody immune response for protection againstthe viral disease.

The adjuvant, which may be administered in conjunction with the vaccineof the present invention, is a substance that increases theimmunological response of the pig to the vaccine. The adjuvant may beadministered at the same time and at the same site as the vaccine, or ata different time, for example, as a booster. Adjuvants also mayadvantageously be administered to the pig in a manner or at a sitedifferent from the manner or site in which the vaccine is administered.Suitable adjuvants include, but are not limited to, aluminum hydroxide(alum), immunostimulating complexes (ISCOMS), non-ionic block polymersor copolymers, cytokines (like IL-1, IL-2, IL-7, IFN-α, IFN-β, IFN-γ,etc.), saponins, monophosphoryl lipid A (MLA), muramyl dipeptides (MDP)and the like. Other suitable adjuvants include, for example, aluminumpotassium sulfate, heat-labile or heat-stable enterotoxin isolated fromEscherichia coli, cholera toxin or the B subunit thereof, diphtheriatoxin, tetanus toxin, pertussis toxin, Freund's incomplete or completeadjuvant, etc. Toxin-based adjuvants, such as diphtheria toxin, tetanustoxin and pertussis toxin may be inactivated prior to use, for example,by treatment with formaldehyde.

The vaccines may further contain additional antigens to promote theimmunological activity of the infectious chimeric PCV DNA clones suchas, for example, porcine reproductive and respiratory syndrome virus(PRRSV), porcine parvovirus (PPV), other infectious swine agents andimmune stimulants.

The new vaccines of this invention are not restricted to any particulartype or method of preparation. The cloned viral vaccines include, butare not limited to, infectious DNA vaccines (i.e., using plasmids,vectors or other conventional carriers to directly inject DNA intopigs), live vaccines, modified live vaccines, inactivated vaccines,subunit vaccines, attenuated vaccines, genetically engineered vaccines,etc. These vaccines are prepared by standard methods known in the art.

The live viral vaccine is generally the most desirable vaccine in thatall possible immune responses are activated in the recipient of thevaccine, including systemic, local, humoral and cell-mediated immuneresponses. A killed vaccine, on the other hand, can only induce humoralimmune response. Albeit the most desirable, however, live viral vaccineshave several disadvantages, such as the potential risk of contaminationwith live adventitious viral agents or the risk that the virus mayrevert to virulence in the field. Remarkably, the unique PCV1-2 chimericDNA of the present invention overcomes those disadvantages. Using onlythe immunogenic genes of the pathogenic PCV2, the chimeric DNAconstructs a live, replicating chimeric virus that is nonpathogenic yetelicits the complete, beneficial immune responses of live viral vaccinesagainst the pathogenic PCV2 virus. The live virus vaccine based on thechimeric virus will have little chance, if any, for reversion to apathogenic phenotype. Thus, the new chimeric virus based on thestructure of the nonpathogenic PCV1 has a huge advantage over anyrecombinant PCV2 DNA virus, any live, attenuated PCV2 vaccine or anyother type of vaccine predicated solely on PCV2 for immunity against thePCV2 infections.

Although the live viral vaccine is most preferred, other types ofvaccines may be used to inoculate pigs with the new chimeric virus andother antigens described herein. To prepare inactivated virus vaccines,for instance, the virus propagation from the infectious DNA clone isdone by methods known in the art or described herein. Serial virusinactivation is then optimized by protocols generally known to those ofordinary skill in the art.

Inactivated virus vaccines may be prepared by treating the chimericvirus derived from the cloned PCV DNA with inactivating agents such asformalin or hydrophobic solvents, acids, etc., by irradiation withultraviolet light or X-rays, by heating, etc. Inactivation is conductedin a manner understood in the art. For example, in chemicalinactivation, a suitable virus sample or serum sample containing thevirus is treated for a sufficient length of time with a sufficientamount or concentration of inactivating agent at a sufficiently high (orlow, depending on the inactivating agent) temperature or pH toinactivate the virus. Inactivation by heating is conducted at atemperature and for a length of time sufficient to inactivate the virus.Inactivation by irradiation is conducted using a wavelength of light orother energy source for a length of time sufficient to inactivate thevirus. The virus is considered inactivated if it is unable to infect acell susceptible to infection.

The preparation of subunit vaccines typically differs from thepreparation of a modified live vaccine or an inactivated vaccine. Priorto preparation of a subunit vaccine, the protective or antigeniccomponents of the vaccine must be identified. Such protective orantigenic components include certain amino acid segments or fragments ofthe viral capsid proteins which raise a particularly strong protectiveor immunological response in pigs; single or multiple viral capsidproteins themselves, oligomers thereof, and higher-order associations ofthe viral capsid proteins which form virus substructures or identifiableparts or units of such substructures; oligoglycosides, glycolipids orglycoproteins present on or near the surface of the virus or in viralsubstructures such as the lipoproteins or lipid groups associated withthe virus, etc. Preferably, a capsid protein, such as the proteinencoded by the ORF2 gene, is employed as the antigenic component of thesubunit vaccine. Other proteins encoded by the infectious DNA clone mayalso be used. These immunogenic components are readily identified bymethods known in the art. Once identified, the protective or antigenicportions of the virus (i.e., the “subunit”) are subsequently purifiedand/or cloned by procedures known in the art. The subunit vaccineprovides an advantage over other vaccines based on the live virus sincethe subunit, such as highly purified subunits of the virus, is lesstoxic than the whole virus.

If the subunit vaccine is produced through recombinant genetictechniques, expression of the cloned subunit such as the ORF2 (capsid)gene, for example, may be optimized by methods known to those in the art(see, for example, Maniatis et al., “Molecular Cloning: A LaboratoryManual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, M A., 1989).If the subunit being employed represents an intact structural feature ofthe virus, such as an entire capsid protein, the procedure for itsisolation from the virus must then be optimized. In either case, afteroptimization of the inactivation protocol, the subunit purificationprotocol may be optimized prior to manufacture.

To prepare attenuated vaccines from pathogenic clones, the tissueculture adapted, live, pathogenic PCV2 is first attenuated (renderednonpathogenic or harmless) by methods known in the art, typically madeby serial passage through cell cultures. Attenuation of pathogenicclones may also be made by gene deletions or viral-producing genemutations. Then, the attenuated PCV2 viruses may be used to constructadditional chimeric PCV1-2 viruses that retain the nonpathogenicphenotype of PCV1 but can vary in the strength of the immunogenicitytraits selected from the PCV2 genome through recombinant technology.Desirably, the attenuation of the PCV2 is accomplished by obtaining theP110A, R191S or both mutations in the ORF2 and using the mutant PCV2 toconstruct the chimeric PCV1-2 viruses as described herein.

The most preferred vaccine employs the live chimeric virus DNA clone, inparticular, the clone containing the immunogenic genes of PCV2 cloned inthe backbone of the nonpathogenic PCV1. Advantageously, the livechimeric virus, which is naturally avirulent when constructed throughgenetic engineering, does not require time-consuming attenuationprocedures. The virus uniquely serves as a live but nonpathogenicreplicating virus that produces immunogenic proteins against PCV2 duringvirus replication, which can then elicit a full range of immuneresponses against the pathogenic PCV2.

As a further benefit, the preferred live chimeric virus of the presentinvention provides a genetically stable vaccine that is easier to make,store and deliver than other types of attenuated vaccines. Avirulent orattenuated vaccines based upon chimeric viruses are generally consideredas safe as, if not safer than, the traditionally modified live vaccines(J. Arroyo et al., “Molecular basis for attenuation of neurovirulence ofa yellow fever Virus/Japanese encephalitis virus chimera vaccine(ChimeriVax-JE),” J. Virol. 75(2):934-942 (2001); F. Guirakhoo et al.,“Recombinant chimeric yellow fever-dengue type 2 virus is immunogenicand protective in nonhuman primates,” J. Virol. 74(12):5477-5485 (2000);S. Tang et al., “Toward a poliovirus-based simian immunodeficiency virusvaccine: correlation between genetic stability and immunogenicity,” J.Virol. 71(10):7841-7850 (1997)). For example, the ChimeriVax-JE vaccineagainst Japanese encephalitis virus (JEV), which is a geneticallyengineered derivative of the yellow fever virus vaccine YFV17D in whichthe genes encoding the structural proteins prM and E of YFV17D arereplaced with the corresponding genes of the attenuated JEV SA14-14-2strain, has been shown to be genetically stable after prolonged passagesboth in vitro and in vivo (J. Arroyo et al., 2001, supra). Anotherchimeric virus vaccine ChimeriVax-D2 against Dengue virus type 2, whichis an attenuated chimeric yellow fever (YF)-dengue type 2 (dengue-2)virus, has also been found to be genetically stable; its sequences werereported to be unchanged after 18 passages in Vero cells (F. Guirakhooet al., 2000, supra).

Another preferred vaccine of the present invention utilizes suitableplasmids for delivering the nonpathogenic chimeric DNA clone to pigs. Incontrast to the traditional vaccine that uses live or killed cellculture propagated whole virus, this invention provides for the directinoculation of pigs with the plasmid DNA containing the infectiouschimeric viral genome.

Additional genetically engineered vaccines, which are desirable in thepresent invention, are produced by techniques known in the art. Suchtechniques involve, but are not limited to, further manipulation ofrecombinant DNA, modification of or substitutions to the amino acidsequences of the recombinant proteins and the like.

Genetically engineered vaccines based on recombinant DNA technology aremade, for instance, by identifying alternative portions of the viralgene encoding proteins responsible for inducing a stronger immune orprotective response in pigs (e.g., proteins derived from ORF3, ORF4,etc.). Such identified genes or immuno-dominant fragments can be clonedinto standard protein expression vectors, such as the baculovirusvector, and used to infect appropriate host cells (see, for example,O'Reilly et al., “Baculovirus Expression Vectors: A Lab Manual,” Freeman& Co., 1992). The host cells are cultured, thus expressing the desiredvaccine proteins, which can be purified to the desired extent andformulated into a suitable vaccine product.

If the clones retain any undesirable natural abilities of causingdisease, it is also possible to pinpoint the nucleotide sequences in theviral genome responsible for any residual virulence, and geneticallyengineer the virus avirulent through, for example, site-directedmutagenesis. Site-directed mutagenesis is able to add, delete or changeone or more nucleotides (see, for instance, Zoller et al., DNA3:479-488, 1984). An oligonucleotide is synthesized containing thedesired mutation and annealed to a portion of single stranded viral DNA.The hybrid molecule, which results from that procedure, is employed totransform bacteria. Then double-stranded DNA, which is isolatedcontaining the appropriate mutation, is used to produce full-length DNAby ligation to a restriction fragment of the latter that is subsequentlytransfected into a suitable cell culture. Ligation of the genome intothe suitable vector for transfer may be accomplished through anystandard technique known to those of ordinary skill in the art.Transfection of the vector into host cells for the production of viralprogeny may be done using any of the conventional methods such ascalcium-phosphate or DEAE-dextran mediated transfection,electroporation, protoplast fusion and other well-known techniques(e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual,” ColdSpring Harbor Laboratory Press, 1989). The cloned virus then exhibitsthe desired mutation. Alternatively, two oligonucleotides can besynthesized which contain the appropriate mutation. These may beannealed to form double-stranded DNA that can be inserted in the viralDNA to produce full-length DNA.

Genetically engineered proteins, useful in vaccines, for instance, maybe expressed in insect cells, yeast cells or mammalian cells. Thegenetically engineered proteins, which may be purified or isolated byconventional methods, can be directly inoculated into pigs to conferprotection against viral infection or postweaning multisystemic wastingsyndrome (PMWS) caused by PCV2.

An insect cell line (like HI-FIVE) can be transformed with a transfervector containing nucleic acid molecules obtained from the virus orcopied from the viral genome which encodes one or more of theimmuno-dominant proteins of the virus. The transfer vector includes, forexample, linearized baculovirus DNA and a plasmid containing the desiredpolynucleotides. The host cell line may be co-transfected with thelinearized baculovirus DNA and a plasmid in order to make a recombinantbaculovirus.

Alternatively, DNA from a pig suffering from PMWS, which encode one ormore capsid proteins, the infectious PCV2 molecular DNA clone or thecloned PCV chimeric DNA genome can be inserted into live vectors, suchas a poxvirus or an adenovirus and used as a vaccine.

An immunologically effective amount of the vaccines of the presentinvention is administered to a pig in need of protection against viralinfection or PMWS. The immunologically effective amount or theimmunogenic amount that inoculates the pig can be easily determined orreadily titrated by routine testing. An effective amount is one in whicha sufficient immunological response to the vaccine is attained toprotect the pig exposed to the virus which causes PMWS. Preferably, thepig is protected to an extent in which one to all of the adversephysiological symptoms or effects of the viral disease are significantlyreduced, ameliorated or totally prevented.

The vaccine can be administered in a single dose or in repeated doses.Dosages may range, for example, from about 1 microgram to about 1,000micrograms of the plasmid DNA containing the infectious chimeric DNAgenome (dependent upon the concentration of the immuno-active componentof the vaccine), preferably 100 to 200 micrograms of the chimeric PCV1-2DNA clone, but should not contain an amount of virus-based antigensufficient to result in an adverse reaction or physiological symptoms ofviral infection. Methods are known in the art for determining ortitrating suitable dosages of active antigenic agent to find minimaleffective dosages based on the weight of the pig, concentration of theantigen and other typical factors. Preferably, the infectious chimericviral DNA clone is used as a vaccine, or a live infectious chimericvirus can be generated in vitro and then the live chimeric virus is usedas a vaccine. In that case, from about 50 to about 10,000 of the 50%tissue culture infective dose (TCID₅₀) of live chimeric virus, forexample, can be given to a pig.

Desirably, the vaccine is administered to a pig not yet exposed to thePCV virus. The vaccine containing the chimeric PCV1-2 infectious DNAclone or other antigenic forms thereof can conveniently be administeredintranasally, transdermally (i.e., applied on or at the skin surface forsystemic absorption), parenterally, etc. The parenteral route ofadministration includes, but is not limited to, intramuscular,intravenous, intraperitoneal, intradermal (i.e., injected or otherwiseplaced under the skin) routes and the like. Since the intramuscular andintradermal routes of inoculation have been successful in other studiesusing viral infectious DNA clones (E. E. Sparger et al., “Infection ofcats by injection with DNA of feline immunodeficiency virus molecularclone,” Virology 238:157-160 (1997); L. Willems et al., “In vivotransfection of bovine leukemia provirus into sheep,” Virology189:775-777 (1992)), these routes are most preferred, in addition to thepractical intranasal route of administration. Although less convenient,it is also contemplated that the vaccine is given to the pig through theintralymphoid route of inoculation. A unique, highly preferred method ofadministration involves directly injecting the plasmid DNA containingPCV1-2 chimera into the pig intramuscularly, intradermally,intralymphoidly, etc.

When administered as a liquid, the present vaccine may be prepared inthe form of an aqueous solution, syrup, an elixir, a tincture and thelike. Such formulations are known in the art and are typically preparedby dissolution of the antigen and other typical additives in theappropriate carrier or solvent systems. Suitable carriers or solventsinclude, but are not limited to, water, saline, ethanol, ethyleneglycol, glycerol, etc. Typical additives are, for example, certifieddyes, flavors, sweeteners and antimicrobial preservatives such asthimerosal (sodium ethylmercurithiosalicylate). Such solutions may bestabilized, for example, by addition of partially hydrolyzed gelatin,sorbitol or cell culture medium, and may be buffered by conventionalmethods using reagents known in the art, such as sodium hydrogenphosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate,potassium dihydrogen phosphate, a mixture thereof, and the like.

Liquid formulations also may include suspensions and emulsions thatcontain suspending or emulsifying agents in combination with otherstandard co-formulants. These types of liquid formulations may beprepared by conventional methods. Suspensions, for example, may beprepared using a colloid mill. Emulsions, for example, may be preparedusing a homogenizer.

Parenteral formulations, designed for injection into body fluid systems,require proper isotonicity and pH buffering to the corresponding levelsof porcine body fluids. Isotonicity can be appropriately adjusted withsodium chloride and other salts as needed. Suitable solvents, such asethanol or propylene glycol, can be used to increase the solubility ofthe ingredients in the formulation and the stability of the liquidpreparation. Further additives that can be employed in the presentvaccine include, but are not limited to, dextrose, conventionalantioxidants and conventional chelating agents such as ethylenediaminetetraacetic acid (EDTA). Parenteral dosage forms must also be sterilizedprior to use.

Another embodiment of the present invention involves a new method ofpreparing an infectious, nonpathogenic chimeric nucleic acid molecule ofPCV1-2, which comprises removing an open reading frame (ORF) gene of anucleic acid molecule encoding an infectious nonpathogenic PCV1,replacing the same position with an immunogenic ORF gene of a nucleicacid molecule encoding an infectious pathogenic PCV2, and recovering thechimeric nucleic acid molecule. The nucleic acid molecule is typicallyDNA. A preferred method replaces the ORF2 gene of the nonpathogenic PCV1DNA with the immunogenic ORF2 capsid gene of the infectious pathogenicmolecular DNA of PCV2 described herein. It is contemplated that otherORF positions or immunogenic fragments thereof can be exchanged betweenthe PCV1 and PCV2 DNA to construct the attenuated infectious chimericDNA clones according to the methods described herein.

The recombinant nucleic acid molecule is then used to construct thelive, infectious, replicating chimeric virus of the present inventionthat advantageously retains the nonpathogenic nature of PCV1 yetexpresses the immunogenic ORF2 protein of the pathogenic PCV2 andelicits a complete immune response against the pathogenic PCV2.Desirably, the PCV1-2 DNA clone serves as a genetically engineeredavirulent, live vaccine against PCV2 infection and PMWS in pigs.

An infectious DNA clone of PCV2 is constructed, as described herein, sothat a biologically pure and homogeneous infectious virus stock can begenerated for pathogenesis studies and the development of nonpathogenic,chimeric vaccines. The course of clinical disease, virus distributionand pathological lesions associated with PCV2 infection are moredefinitively characterized by using this molecular DNA clone and abiologically pure and homogeneous infectious PCV2 virus stock derivedfrom the molecular DNA clone than have been observed in the past, whichlends itself to the development of the desired vaccine products of thepresent invention.

The PCV2 molecular clone is generated by ligating two copies of thecomplete PCV2 genome in tandem into the pSK vector. In sharp contrast tothe single copy genome disclosed in the art, the infectious DNA PCV2clone made by the methods described herein contains two complete copiesof the PCV2 genome ligated together in tandem repeat. Ligating twocopies of genome in tandem provides a similar circular genome thatmimics the usual circular genome of PCV2. The advantage of having twocopies of the genome in tandem in the infectious DNA PCV2 clone is to beable to maximize replication when the infectious DNA clone istransfected in vitro and in vivo. Thus, the clone of the inventionoperates more efficiently and effectively than the prior single copygenome.

Infection of animals with the molecular viral clone is extremely usefulto studying the genetic determinants of viral replication and virulencein the host. Type-2 porcine circovirus (PCV2) has been incriminated asthe causative agent of postweaning multisystemic wasting syndrome(PMWS). PMWS is a complex disease syndrome in swine and multiple factorsmay be involved in the clinical presentation of PMWS. However, thedifficulty in producing a biologically pure form of PCV2 due to thepresence of other common swine agents in the tissue homogenates ofdiseased pigs has impeded a definitive characterization of the clinicaldisease and pathological lesions solely attributable to PCV2 infection.This is the first time an infectious molecular DNA clone of PCV2 hasbeen constructed and used to characterize the disease and pathologicallesions associated with PCV2 infection by direct in vivo transfection ofpigs with the molecular clone.

The homogeneous PCV2 live virus stock derived from the molecular cloneis shown to be infectious in vitro when transfected into PK-15 cells.The cloned PCV2 genomic DNA is also infectious when directly injectedinto the livers and superficial iliac lymph nodes ofspecific-pathogen-free (SPF) pigs. Animals injected with the cloned PCV2plasmid DNA develop an infection and disease resembling that induced byintranasal inoculation with a homogenous, infectious PCV2 live virusstock. Seroconversion to PCV2-specific antibody is detected in themajority of pigs from the inoculated groups at 35 days postinoculation(DPI).

The onset and duration of viremia in pigs inoculated with the chimericPCV1-2 DNA clone are similar to those of the pigs inoculated with thenonpathogenic PCV1 DNA clone, whereas viremia in pigs inoculated withthe PCV2 clone appears earlier and lasted longer. Beginning at 14 DPIand lasting about 2-4 weeks, viremia is detected in the majority of thePCV2-inoculated animals. Similarly, the majority of inoculated pigsnecropsied at 35 DPI seroconverted to PCV2-antibodies. PCV2 antigen isdetected in various tissues and organs in inoculated pigs. Gross lesionsare limited to the lungs and lymph nodes, and are characterized bysystematically enlarged tan colored lymph nodes, lungs that failed tocollapse and mild multifocal tan-colored foci of consolidation. Grosslesions affecting the lymph nodes in both the nonpathogenic PCV1 and thechimeric PCV1-2 inoculated pigs are mild and limited to only a fewanimals, whereas the pathogenic PCV2 inoculated pigs all havemoderate-to-severe swelling and discoloration of lymphoid tissues (Table9, below). Statistical analysis reveals that the scores of the grosslesions in the lymph nodes of the chimeric PCV1-2 inoculated animals aresimilar to those in nonpathogenic PCV1 inoculated pigs. At 21 DPI, PCV2inoculated pigs have gross lesions that are statistically more severethan those of the PCV1 and the chimeric PCV1-2 inoculated pigs.Histopathological lesions and PCV2-specific antigen are detected innumerous tissues and organs including brain, lung, heart, kidney,tonsil, lymph nodes, spleen, ileum and liver of the inoculated(infected) pigs. The histopathological lesions in multiple tissues andorgans similar to those of PMWS are reproduced with the PCV2 molecularDNA clone as well as with the infectious virus prepared in vitro fromthe molecular DNA clone. Microscopically, at both 21 and 49 DPIs, thechimeric PCV1-2 inoculated animals have statistically less microscopiclesions than the PCV2 inoculated animals. The microscopic lesion scoresin lymph nodes of the chimeric PCV1-2 inoculated pigs are similar tothose of the nonpathogenic PCV1, the reciprocal chimeric PCV2-1 and theuninoculated control animals. Moderate to severe microscopic lesions arefound in multiple tissues of pathogenic PCV2 inoculated animalsincluding lung, liver, lymphoid, spleen, brain, heart, kidney and tonsiltissue. However, in chimeric PCV1-2 inoculated animals, mild to moderatemicroscopic lesions are limited only to liver, lymph nodes and kidneytissues (see Table 10, below).

There are no remarkable clinical signs of PMWS in the control or any ofthe inoculated pigs. Although the characteristic clinical symptoms ofPMWS are not observed with the cloned PCV2 plasmid DNA (the infectiousPCV2 DNA clone) or with a biologically pure PCV2 infectious virus stock,PCV2 is clearly responsible for the PMWS-like histopathological lesionsreproduced in the below illustrative examples. It is generally believedthat PCV2 is the primary but not the sole pathogenic agent responsiblefor the onset of clinical PMWS.

This invention more definitively characterizes the clinical course andpathological lesions exclusively attributable to PCV2 infection. Thepresent data in the below illustrative examples indicate that thereadily reproduced, cloned PCV2 genomic DNA is available to replaceinfectious virus for the PCV2 pathogenesis and immunization studies.While PCV2 is shown as essential for development of PMWS, other factorsor agents such as PRRSV, PPV, etc. may be required to induce the fullspectrum of clinical signs and lesions associated with advanced cases ofPMWS. However, with the knowledge that PCV2 is a key factor, the novelinfectious, replicating viral clone of the present invention can befurther modified or genetically engineered to achieve the desiredoptimal immunogenic effect through methods known to those of ordinaryskill in immunology and molecular genetics.

The availability of the infectious DNA clone of PCV2 described hereinmakes it feasible to develop the genetically engineered attenuatedvaccine for preventing PCV2 infection and PMWS in pigs. It is known thatPCV2 replicates in the lymph nodes, lungs and liver during naturalinfection, and one of the major pathogenic effects is the impairment ofthe immune system by degradation of the lymphoid structures (S. Krakowkaet al., 2001, supra; G. M. Allan and J. A. Ellis, 2000, supra; S.Kennedy et al., 2000, supra; G. J. Wellenberg et al., 2000, supra; G. M.Allan et al., “Experimental reproduction of severe wasting disease byco-infection of pigs with porcine circovirus and porcine parvovirus,” J.Comp. Pathol. 121:1-11 (1999); J. Ellis et al., “Reproduction of lesionsof postweaning multisystemic wasting syndrome in gnotobiotic piglets,”J. Vet. Diagn. Invest. 11:3-14 (1999); J. C. Harding and E. G. Clark,1997, supra). By using this novel infectious PCV2 molecular DNA clone,the clinical disease, pathological lesions and virus distributionexclusively attributable to PCV2 infection are more definitivelycharacterized.

The structural and functional relationships of the PCV genes are betterunderstood due to the availability of the PCV2, PCV1, chimeric PCV1-2,and reciprocal chimeric PCV2-1 infectious DNA clones described herein.Will et al., “Cloned HBV DNA causes hepatitis in chimpanzees,” Nature299:740-742 (1982), first demonstrated the feasibility of using a clonedhepatitis B virus DNA to infect chimpanzees by direct in vivo injection.This approach has since been used to study viral replication andpathogenesis of several other viruses (T. W. Dubensky et al., “Directtransfection of viral and plasmid DNA into the liver or spleen of mice,”Proc. Natl. Acad. Sci. USA 81:7529-7533 (1984); R. Girones et al.,“Complete nucleotide sequence of a molecular clone of woodchuckhepatitis virus that is infectious in the natural host,” Proc. Natl.Acad. Sci. USA 86:1846-1849 (1989); N. L. Letvin et al., “Risks ofhandling HIV,” Nature 349:573 (1991); C. Seeger et al., “The clonedgenome of ground squirrel hepatitis virus is infectious in the animal.Proc. Natl. Acad. Sci. USA. 81:5849-5852 (1984); E. E. Sparger et al.,“Infection of cats by injection with DNA of feline immunodeficiencyvirus molecular clone,” Virology 238:157-160 (1997); R. Sprengel et al.,“Homologous recombination between hepadnaviral genomes following in vivoDNA transfection: implications for studies of viral infectivity,”Virology 159:454-456 (1987); H. Will et al., 1982, supra; L. Willems etal., “In vivo transfection of bovine leukemia provirus into sheep,”Virology 189:775-777 (1992)).

The construction of an infectious PCV2 molecular DNA clone, and thedemonstration of infection by direct injection of cloned PCV2 plasmidDNA into the liver and lymph nodes of pigs in the context of the presentinvention are advantageous for PCV2 studies. This in vivo transfectionsystem will enhance the study of the structural and functionalrelationship of PCV2 genes using recombinant plasmids constructed invitro to test different regions or genes of PCV2 for their roles invirus replication and pathogenesis in the host. The replication andpathogenesis of PCV2 can be studied in vivo without having to produceinfectious virus stocks by propagating PCV2 in cell cultures. This isadvantageous as serial cell culture passages may select for viralvariants. Another advantage of using cloned PCV2 genomic DNA, instead oflive virus, for animal studies is its relative ease for quantitation ofthe inoculation dose. The amount of the cloned PCV2 DNA used for animalinoculation can be easily determined by a spectrophotometer, whereas thedose of live PCV2 virus requires infectivity titration in cell culturesand confirmation of infection by IFA. Direct injection of animals withcloned PCV2 plasmid DNA eliminates the problems associated with thepresence of other indigenous swine agents in tissue homogenate inoculain animal studies.

In the present invention, the immunogenic ORF2 capsid gene is switchedbetween the pathogenic PCV2 and the nonpathogenic PCV1 to produce theunique structure of the chimeric PCV1-2 infectious DNA clone.Surprisingly and advantageously, the chimeric PCV1-2 infectious clonereplicated, expressed the immunogenic ORF2 capsid antigen in vitro andin vivo, and induced a specific antibody response against PCV2 ORF2 butretained the nonpathogenic nature of PCV1. The chimeric PCV1-2infectious DNA clone has the ability to induce a strong immune responseagainst PCV2 while inducing only a limited infection with mildpathologic lesions similar to that of the nonpathogenic PCV1. Forvaccine development, the relatively easy storage and stability of clonedDNA, and the economy of large-scale recombinant PCV2 plasmid DNA andchimeric PCV1-2 DNA clone production provides an attractive means ofdelivering a live, infectious viral DNA vaccine or geneticallyengineered, attenuated viral vaccines to pigs. Therefore, the chimericPCV1-2 infectious DNA clone taught in this invention is a useful vaccinecandidate against PCV2 infection and PMWS.

It should be appreciated that all scientific and technological termsused herein have the same meaning as commonly understood by those ofordinary skill in the art. For purposes of this invention, the term“infectious” means that the virus replicates in pigs, regardless ofwhether or not the virus causes any diseases. “SPF” refers toSpecific-pathogen-free pigs. The “gnotobiotic” pigs intend germ-freepigs. The terms “PCV2 plasmid DNA,” “PCV2 genomic DNA” and “PCV2molecular DNA” are being used interchangeably to refer to the samecloned nucleotide sequence.

The infectious PCV1/PCV2 chimeric DNA clone (strain designation “PCV1-2chimera”), the infectious PCV2 molecular DNA clone (strain designation“PCV2 clone”) and the biologically pure and homogeneous PCV2 stockderived from an Iowa sample of PCV2 that had been isolated from a pigwith severe PMWS and identified as isolate number 40895 (straindesignation “PCV2 #40895”) are deposited under the conditions mandatedby 37 C.F.R. § 1.808 and maintained pursuant to the Budapest Treaty inthe American Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209, U.S.A. The DNA sequences described herein arecontained within 6,490 bp plasmids cloned into pBluescript SK(+) vector(pSK) (Stratagene Inc., La Jolla, Calif.) and transformed intoEscherichia coli DH5a competent cells. The plasmids containing theinfectious chimeric PCV1-2 DNA clone (identified as “chimeric porcinecircovirus Type 1 (PCV1) and Type 2 (PCV2) infectious DNA clone”) andthe infectious PCV2 molecular DNA clone (identified as “infectious DNAclone of Type 2 porcine circovirus (PCV2)”) have been deposited in theATCC on Dec. 7, 2001 and have been assigned ATCC Patent DepositDesignations PTA-3912 and PTA-3913, respectively. It should beappreciated that other plasmids, which may be readily constructed usingsite-directed mutagenesis and the techniques described herein, are alsoencompassed within the scope of the present invention. The biologicallypure and homogeneous PCV2 sample of isolate number 40895 (identified as“Type 2 porcine circovirus (PCV2)”) has also been deposited in the ATCCon Dec. 7, 2001 and has been assigned ATCC Patent Deposit DesignationPTA-3914. The genomic (nucleotide) sequence of the PCV2 isolate number40895 has been deposited with the Genbank database and has been publiclyavailable since Jul. 23, 2000 under accession number AF264042.

The following examples demonstrate certain aspects of the presentinvention. However, it is to be understood that these examples are forillustration only and do not purport to be wholly definitive as toconditions and scope of this invention. It should be appreciated thatwhen typical reaction conditions (e.g., temperature, reaction times,etc.) have been given, the conditions both above and below the specifiedranges can also be used, though generally less conveniently. Theexamples are conducted at room temperature (about 23° C. to about 28°C.) and at atmospheric pressure. All parts and percents referred toherein are on a weight basis and all temperatures are expressed indegrees centigrade unless otherwise specified.

A further understanding of the invention may be obtained from thenon-limiting examples that follow below.

Example 1 Generation of a PK-15 Cell Line Free of PCV1 Contamination

The source of the PCV2 isolate was from a spleen tissue sample of a pigwith naturally occurring PMWS (PCV2 serial identification number 40895,referred to as “isolate 40895”) (M. Fenaux et al., 2000, supra).Immunohistochemical staining (IHC) with PCV2-specific antibody confirmedthe presence of PCV2 antigen in the tissue. The spleen tissues werestored at −80° C. until use.

The PK-15 cell line purchased from the American Type Culture Collection(ATCC accession number CCL-33) was persistently infected with PCV1 (G.C. Dulac and A. Afshar, 1989, supra). Since only a subpopulation ofPK-15 cells was persistently infected (id.), a PK-15 cell line that isfree of PCV1 contamination by end-point dilution was generated. Protocolproceeded as follows: PK-15 cells were grown in MEM with Earle's saltsand L-glutamine (Life Technologies, Inc., Grand Island, N.Y.)supplemented with 10% fetal bovine serum (FBS) and 1× antibiotic (LifeTechnologies, Inc.). Confluent cell monolayers were trypsinized, and thecells were then counted and serially diluted to an end point with onecell per 0.2 ml. The end point dilution was plated in 96-well plates andallowed to grow into a monolayer starting from a single cell. Cells fromeach well were tested for PCV1 DNA using a PCR-RFLP assay capable ofdetecting and differentiating PCV1 and PCV2 (M. Fenaux et al., 2000,supra). PK-15 cells from wells that were tested negative for PCV1 by thePCR-RFLP assay were subsequently expanded. The PCV1 free PK-15 cell linewas subcultured five additional passages and was found negative for PCV1DNA by PCR at each passage.

Four cell lines that were negative for PCV1 contamination were producedby the end-point dilution of the persistently infected PK-15 cells fromATCC. The cell lines remained negative for PCV1 by PCR after the fiveadditional passages. One of the cell lines was subsequently expanded andwas shown to be able to support PCV2 replication when the cells weretransfected with the PCV2 molecular DNA clone (FIG. 2) and infected withPCV2 virus. The cloned cells were further used for the in vitrotransfection of PCV2 molecular DNA clone to generate a biologically purePCV2 infectious virus stock for the animal inoculation experiment.

Example 2 Construction of the PCV2 Infectious DNA Clone

To construct a PCV2 molecular DNA clone, a pair of PCR primers wasdesigned according to the published sequence of the PCV2 isolate 40895(M. Fenaux et al., 2000, supra): forward primer F-PCVSAC2(5′-GAACCGCGGGCTGGCTGAACTTTTGAAAGT-3′), set forth in SEQ ID NO:5, andreverse primer R-PCVSAC2 (5′-GCACCGCGGAAATTTCTGACAAACGTTACA-3′), setforth in SEQ ID NO:6. This pair of primers amplifies the complete genomeof PCV2 with an overlapping region containing the unique SacIIrestriction enzyme site (FIG. 1). DNA was extracted using the QIAamp DNAMinikit (Qiagen, Inc., Valencia, Calif.) from a spleen tissue sample ofa pig with naturally occurring PMWS (isolate 40895) (M. Fenaux et al.,2000, supra). The extracted DNA was amplified by PCR with AmpliTaq Goldpolymerase (Perkin-Elmer, Norwalk, Conn.). The PCR reaction consisted ofan initial enzyme activation step at 95° C. for 9 min, followed by 35cycles of denaturation at 94° C. for 1 min, annealing at 48° C. for 1min, extension at 72° C. for 3 min, and a final extension at 72° C. for7 min. The PCR product of expected size was separated by gelelectrophoresis and purified with the glassmilk procedure with aGeneclean Kit (Bio 101, Inc., La Jolla, Calif.).

To construct a molecular DNA clone containing a tandem dimer of PCV2genome, the PCR product containing the complete PCV2 genome was firstligated into the advanTAge plasmid vector (Clontech, Palo Alto, Calif.).E. Coli DH5a competent cells were transformed. The recombinant plasmidswere verified by restriction enzyme digestion. The full length PCV2genomic DNA was excised from the advanTAge vector by digestion withSacII restriction enzyme. The digested PCV2 genomic DNA was ligated withT4 DNA ligase at 37° C. for only 10 min, which favors the production oftandem dimers. The tandem dimers were subsequently cloned intopBluescript SK(+) vector (pSK) (Stratagene Inc., La Jolla, Calif.) (FIG.1). Recombinant plasmids containing tandem dimers of PCV2 genome (hereinreferred to as PCV2 molecular DNA clone) were confirmed by PCR,restriction enzyme digestion, and DNA sequencing. The DNA concentrationof the recombinant plasmids was determined spectrophotometrically.

Specifically, the complete genome of the PCV2 (isolate 40895) wasamplified by PCR to construct the infectious PCV2 molecular DNA clone.Two copies of the complete PCV2 genome were ligated in tandem into thepSK vector to produce the PCV2 molecular DNA clone (FIG. 1). Theinfectivity of the PCV2 molecular DNA clone was determined by in vitrotransfection of the PK-15 cells. IFA with PCV2-specific antibodyconfirmed that the molecular DNA clone is infectious in vitro and thatabout 10-15% of the PK-15 cells were transfected. PCV2-specific antigenwas visualized by IFA in the nucleus, and to a lesser degree, cytoplasmof the transfected cells (FIG. 2). The cells mock-transfected with theempty pSK vector remained negative for PCV2 antigen.

Example 3 In Vitro Transfection with the PCV2 Molecular DNA Clone andGeneration of a Biologically Pure and Homogenous PCV2 Infectious VirusStock

To test the infectivity of the molecular DNA clone in vitro, PK-15 cellsfree of PCV1 contamination were grown in 8-well LabTek chamber slides.When the PK-15 cells reached about 85% confluency, cells weretransfected with the molecular DNA clone using Lipofectamine PlusReagents according to the protocol supplied by the manufacturer (LifeTechnologies, Inc). Mock-transfected cells with empty pSK vector wereincluded as controls. Three days after transfection, the cells werefixed with a solution containing 80% acetone and 20% methanol at 4° C.for 20 min., and an immunofluorescence assay using a PCV2-specificrabbit polyclonal antisera was performed to determine the in vitroinfectivity of the molecular DNA clone (see below).

To generate a biologically pure and homogeneous PCV2 infectious virusstock for the animal inoculation experiment, PK-15 cells free of PCV1contamination were cultivated in T-25 culture flasks and transfectedwith the PCV2 molecular DNA clone. PK-15 cells were grown to about 85%confluency in T-25 flasks. The cells were washed once with sterile PBSbuffer before transfection. For each transfection reaction in a T-25flask, 12 μg of the PCV2 plasmid DNA was mixed with 16 μl of PlusReagent in 0.35 ml of MEM media. A flask of mock-transfected cells withempty pSK vector was included as the negative control. After incubationat room temperature for 15 min., 50 μl of Lipofectamine Reagent dilutedin 0.35 ml of MEM media was added to the mixture and incubated at roomtemperature for another 15 min. The transfection mixture was then addedto a T-25 flask of PK-15 cells containing 2.5 ml of fresh MEM. Afterincubation at 37° C. for 3 hrs, the media was replaced with fresh MEMmedia containing 2% FBS and 1× antibiotics. The transfected cells wereharvested at 3 days post transfection and stored at −80° C. until use.The infectious titer of the virus stock was determined by IFA (seebelow).

Basically, biologically pure and homogenous PCV2 infectious virus stockwas generated by transfection of PK-15 cells with the PCV2 molecular DNAclone. PCV2 virions produced by in vitro transfection were infectioussince the transfected cell lysates were successfully used to infectPK-15 cells. Thus, the PCV2 molecular DNA clone is capable of producinginfectious PCV2 virions when transfected in vitro. The infectious titerof the homogenous PCV2 virus stock prepared from transfected cells wasdetermined to be 1×10^(4.5) TCID₅₀/ml. This virus stock was used toinoculate pigs in Group 2. Lysates of cells mock-transfected with theempty pSK vector were unable to infect PK-15 cells.

Example 4 Virus Titration by Immunofluorescence Assay (IFA)

To determine the infectious titer of the homogenous PCV2 virus stock,PK-15 cells were cultivated on 8-well LabTek chamber slides. The virusstock was serially diluted 10-fold in MEM, and each dilution wasinoculated onto 10 wells of the monolayers of the PK-15 cells growing onthe LabTek chamber slides. Wells of non-inoculated cells were includedas controls. The infected cells were fixed at 3 days post inoculationwith a solution containing 80% acetone and 20% methanol at 4° C. for 20min. After washing the cells with PBS buffer, the infected cells wereincubated with a 1:1,000 diluted PCV2-specific rabbit polyclonalantibody (S. D. Sorden et al., “Development of apolyclonal-antibody-based immunohistochemical method for the detectionof type 2 porcine circovirus in formalin-fixed, paraffin-embeddedtissue,” J. Vet. Diagn. Invest. 11:528-530 (1999)) at 37° C. for 1 hr.The cells were then washed three times with PBS buffer, and incubatedwith a secondary FITC-labeled goat anti-rabbit IgG (Kirkegaard & PerryLaboratories Inc, Gaithersburg, Md.) at 37° C. for 45 min. After washingthe slides three times with PBS buffer, and the slides were mounted withfluoromount-G, cover-slipped and examined under a fluorescencemicroscope. The 50% tissue culture infectious dose per ml (TCID₅₀/ml)was calculated. Initially, cells were transfected with a plasmidconstruct containing a single copy of PCV2 genome but the infectiousPCV2 titer from the single genome construct is much lower than the onecontaining the tandem genome. Therefore, the plasmid constructcontaining the dimeric form of PCV2 genome was used for the in vitro andin vivo transfection experiments.

Example 5 In Vivo Transfection of Pigs with the PCV2 Molecular DNA Cloneand Experimental Inoculation of Pigs with the Homogeneous PCV2Infectious Virus Stock

Forty specific-pathogen-free (SPF) swine of 4 weeks of age were randomlyassigned into 4 rooms of 10 animals each. Prior to inoculation, the SPFpigs were tested for antibodies to PCV, PRRSV, PPV and swine hepatitis Evirus. Pigs in Group 1 were uninoculated and served as negativecontrols. Pigs in Group 2 were each inoculated intranasally with about1.9×10⁵ TCID₅₀ of the PCV2 infectious virus stock derived from the PCV2molecular DNA clone. Pigs in Group 3 received direct intrahepaticinjection of the recombinant plasmid DNA of the PCV2 molecular clone.Each pig was injected with a total of 200 μg of recombinant plasmid DNA(the cloned PCV2 plasmid DNA), through an ultrasound-guided technique,into 6 different sites of the liver. Pigs in Group 4 were each injectedwith a total of 200 μg of the recombinant PCV2 plasmid DNA directly intothe superficial iliac lymph nodes, and each lymph node received twoseparate injections. The animals were monitored daily for clinical signsof disease. Serum samples were collected from each animal at 0, 7, 14,21, 28, 35 days post inoculation (DPI). At 21 DPI, five pigs wererandomly selected from each group and necropsied. The remaining fiveanimals in each group were necropsied at 35 DPI. Various tissues andorgans were collected during necropsy and processed for histologicalexamination and immunohistochemical staining (see below).

The results are shown in Table 1 below. All inoculated pigs from Groups2, 3 and 4 were negative for PCV2 antibodies at 0 DPI. Two pigs in theuninoculated control Group 1 had detectable PCV2 maternal antibody at 0DPI. The maternal antibody in these two piglets waned by 7 DPI. Noseroconversion to PCV2 antibody was detected in any of the 10uninoculated control pigs. In Group 2 pigs intranasally inoculated withPCV2 infectious virus, 1 piglet seroconverted to PCV2 antibody at 21DPI. By 35 DPI, 4 of the 5 remaining Group 2 pigs had seroconverted.Seroconversion in transfected animals from Groups 3 and 4 first appearedat 28 DPI. By 35 DPI, 5 of 5 remaining pigs from Group 3 and 3 of 5remaining pigs from Group 4 had seroconverted to PCV2 antibody.

PPV antibodies were tested at 3 and 21 DPI for all pigs, and at 35 DPIfor the remaining pigs. Maternal antibodies to the ubiquitous swineagent PPV were detected in the SPF piglets. The PPV HI antibody titersin all piglets but one decreased significantly from 3 DPI (an averagetiter of 1:2,665) to 21 DPI (an average titer of 1:246), indicating theantibody detected in these piglets was passively derived. One piglet hada slightly increased PPV HI titer from 1:32 at 3 DPI to 1:64 at 21 DPI,which is likely due to testing variation. Serum samples collected fromall pigs at 0, 21, and 35 DPI were further tested for PPV DNA with apublished PCR assay (J. M. Soucie et al., “Investigation of porcineparvovirus among persons with hemophilia receiving Hyate: C porcinefactor VIII concentrate,” Transfusion 40:708-711 (2000)). No PPV viremiawas detected from any pigs at any DPI, further indicating the pigs werenot infected by PPV.

TABLE 1 Seroconversion to PCV2 Specific Antibodies in Pigs InoculatedWith PCV2 Live Virus or Directly Injected With Cloned PCV2 Plasmid DNARoute of Days Postinoculation Group Inocula Inoculation 0 7 14 21 28 351 None 2/10^(a) 0/10 0/10 0/10 0/5 0/5 2 PCV2 live virus^(b) Intranasal0/10 0/10 0/10 1/10 1/5 4/5 3 PCV2 DNA^(c) Intrahepatic 0/10 0/10 0/100/10 1/5 5/5 4 PCV2 DNA^(c) Intralymphoid 0/10 0/10 0/10 0/10 1/5 3/5^(a)PCV2 antibody was measured with an ELISA, number positive/numbertested. ^(b)A biologically pure and homogeneous PCV2 virus stockgenerated by transfection of PK-15 cells with PCV2 molecular DNA clone.^(c)Cloned PCV2 genomic DNA in pSK plasmid.

Example 6 PCR-RFLP Analyses

To measure PCV2 viremia in pigs transfected with PCV2 molecular DNAclone and in pigs infected with PCV2 infectious virus stock, serumsamples collected at different DPIs were tested for the presence of PCV2DNA by the general methods of a PCR-RFLP assay previously described (M.Fenaux et al., 2000, supra). Viral DNA was extracted from 50 μl of eachserum sample using the DNAzol® reagent according to the protocolsupplied by the manufacturer (Molecular Research Center, Cincinnati,Ohio). The extracted DNA was resuspended in DNase-, RNase-, andproteinase-free water and tested for PCV2 DNA by PCR-RFLP (id.). PCRproducts from selected animals were sequenced to verify the origin ofthe virus infecting pigs.

Serum samples were collected from all control and inoculated animals at0, 7, 14, 21, 28, and 35 DPIs and assayed for PCV2 viremia by detectionof PCV2 DNA (id.). The results are shown in Table 2 below. PCV2 DNA wasnot detected in the Group 1 uninoculated control pigs at any DPI.Viremia was detected in 7/10 pigs from Group 2 at 14 DPI and 8/10 by 35DPI. Viremia lasted only a few weeks as the PCV2 DNA was not detectableat 28 DPI and 35 DPI in all 5 remaining pigs from Group 2. In Group 3pigs that were intrahepatically injected with PCV2 molecular DNA clone,8/10 pigs were viremic at 14 DPI, and 9/10 pigs had detectable viremiaby 35 DPI. Group 4 pigs were injected with PCV2 molecular DNA clone intothe lymph nodes. Two of 10 pigs at 14 DPI and 8 of 10 pigs at 21 DPIfrom Group 4 were viremic. The results show that PCV2 molecular DNAclone is infectious when injected directly into the liver andsuperficial iliac lymph nodes of SPF pigs. PCR products amplified fromselected animals were sequenced. The sequence of the PCR productsamplified from selected animals was identical to the correspondingregion of the PCV2 molecular DNA clone.

TABLE 2 Detection of Viremia (PCV2 DNA) by PCR in Sera of Inoculated andControl Pigs Route of Days Postinoculation Group Inocula Inoculation 0 714 21 28 35 Total 1 None 0/10^(a) 0/10 0/10 0/10 0/5 0/5 0/10 2 PCV2live virus^(b) Intranasal 0/10 0/10 7/10 5/10 0/5 0/5 8/10 3 PCV2DNA^(c) Intrahepatic 0/10 0/10 8/10 6/10 3/5 3/5 9/10 4 PCV2 DNA^(c)Intralymphoid 0/10 0/10 2/10 8/10 2/5 0/5 8/10 ^(a)10 pigs in eachgroup, number positive/number tested. ^(b)A biologically pure andhomogeneous PCV2 virus stock generated by transfection of PK-15 cellswith PCV2 molecular DNA clone. ^(c)Cloned PCV2 genomic DNA in pSKplasmid.

Example 7 Clinical Evaluation

Pigs were weighed on 0 DPI and at the time of necropsy. Rectaltemperatures and clinical respiratory disease scores, ranging from 0 to6 (0=normal, 6=severe) (P. G. Halbur et al., “Comparison of thepathogenicity of two U.S. porcine reproductive and respiratory syndromevirus isolates with that of the Lelystad virus,” Vet. Pathol. 32:648-660(1995)), were recorded every other day from 0 to 35 DPI. Clinicalobservations including evidence of central nervous system disease, liverdisease (icterus), musculoskeletal disease, and changes in bodycondition, were also recorded daily.

To evaluate the gross pathology and histopathology, five pigs from eachgroup were randomly selected for necropsies at 21 and 35 DPI. Thenecropsy team was blinded to infection status of the pigs at necropsy.Complete necropsies were performed on all pigs. An estimated percentageof the lung with grossly visible pneumonia was recorded for each pigbased on a previously described scoring system (id). The scoring systemis based on the approximate volume that each lung lobe contributes tothe entire lung: the right cranial lobe, right middle lobe, cranial partof the left cranial lobe, and the caudal part of the left cranial lobeeach contribute 10% of the total lung volume, the accessory lobecontributes 5%, and the right and left caudal lobes each contribute27.5%. Other lesions such as enlargement of lymph nodes were notedseparately. Sections for histopathologic examination were taken fromnasal turbinate, lungs (seven sections) (id.), heart, brain, lymph nodes(tracheobronchial, iliac, mesenteric, subinguinal), tonsil, thymus,liver, gall bladder, spleen, joints, small intestine, colon, pancreas,and kidney. The tissues were examined in a blinded fashion and given asubjective score for severity of lung, lymph node, and liver lesions.Lung scores ranged from 0 (normal) to 3 (severe lymphohistiocyticinterstitial pneumonia). Liver scores ranged from 0 (normal) to 3(severe lymphohistiocytic hepatitis). Lymph node scores were for anestimated amount of lymphoid depletion of follicles ranging from 0(normal or no lymphoid depletion) to 3 (severe lymphoid depletion andhistiocytic replacement of follicles).

The serology protocol involved collecting blood on arrival at 11 to 12days of age, and from all pigs at 0, 7, 14, 21, 28, and 35 DPIs. Serumantibodies to PRRSV were assayed using Herd Check PRRSV ELISA (IDEXXLaboratories, Westbrook, Mass.). Serum antibodies to PPV were detectedby a hemagglutination inhibition (HI) assay (H. S. Joo et al., “Astandardized haemagglutination inhibition test for porcine parvovirusantibody,” Aust. Vet. J. 52:422-424 (1976)). Serum antibodies to PCV2were detected by a modified indirect ELISA based on the recombinant ORF2protein of PCV2 (P. Nawagitgul et al., “Modified indirect porcinecircovirus (PCV) type 2-based and recombinant capsid protein(ORF2)-based ELISA for the detection of antibodies to PCV,” Immunol.Clin. Diagn. Lab Immunol. 1:33-40 (2002)). A partially purified PCV2antigen was prepared from Hi Five cells (Invitrogen, Carlsbad, Calif.)infected with recombinant baculovirus containing the major capsid ORF2protein of PCV2 (P. Nawagitgul et al., “Open reading frame 2 of porcinecircovirus type 2 encodes a major capsid protein,” J. Gen. Virol.81:2281-2287 (2000)). Cell lysates of Hi Five cells infected withwild-type baculovirus were prepared similarly and served as negativecontrol antigen. The Immulon 2 HB polystyrene microtiter plates (DynexTechnologies Inc, Chantilly, Va.) were coated with optimalconcentrations of positive and negative antigens at 4° C. for 36 hrs.One hundred μl of each serum sample diluted 1:100 in 5% milk diluent(Kirkegaard & Perry Laboratories, Inc.) was added into each well. Theserum samples were tested in quadruplicate: 2 wells for negative controlantigen and 2 parallel wells for PCV2 antigen. Positive control andnegative control sera were included in each plate. The sera wereincubated at 37° C. for 30 min. and then washed 5 times with 0.1 M PBSbuffer containing 0.1% Tween-20. A peroxidase-labeled secondaryanti-swine IgG (Sigma Co, St. Louis, Mo.) was incubated at 37° C. for 30min. The plates were washed again and incubated with2,2′-azino-di-(3-ethylbenzthiazoline-6-sulfonate) (Kirkegaard & PerryLaboratories Inc) at 37° C. for 15 min. for color development. Theoptical density (OD) was read at 405 nm. The corrected OD of each testedand control sera was calculated by subtraction of mean OD value of thewells containing negative antigen from that of the parallel wellscontaining PCV2 antigen. The data was normalized by dividing thecorrected OD value of a tested serum sample (S) with that of thepositive control serum (P) and reported as S/P ratios. The samples withS/P ratios ≤0.12, 0.12 to 0.2, and >0.2 were considered as negative,equivocal and positive, respectively.

From the results of the clinical evaluation, none of the control andinoculated pigs showed obvious signs of disease resembling those ofclinical PMWS. There was no difference in weight gain or mean rectaltemperatures between any of the four groups. The control pigs of Group 1remained normal throughout the experiment. There was mild transientrespiratory disease observed in the majority of the pigs in PCV2DNA-transfected and PCV2 virus-infected groups from 8 to 14 DPI. Thiswas characterized by mild dyspnea (clinical respiratory scores of 1 to2) of one-to-two days duration in individual pigs and 5-6 days durationfor the group.

There were no gross lesions observed in the control pigs at necropsy.Pigs in the three inoculated groups had gross lesions limited to thelungs and lymph nodes (see Table 3, below). The lesions were similaramong pigs in the PCV2 plasmid DNA-transfected and PCV2 virus-infectedgroups. Lungs failed to collapse and had random, multifocal, moderatelywell-demarcated areas of tan-to-purple consolidation involving 0-2% ofthe lung (FIG. 3) at 21 DPI, and 0-13% of the lung at 35 DPI. Lymphnodes were systemically enlarged 2 to 5 times normal size, firm, and tan(FIG. 3) at both 21 and 35 DPI in most of the pigs from all threePCV2-inoculated groups.

Microscopic examination revealed no lesions in any tissues of thecontrol pigs except for the livers. Eight of ten control pigs had verymild multifocal lymphoplasmacytic inflammation predominately in theperiportal regions of the liver as is commonly observed in normal pigsand considered normal background (P. G. Halbur et al., 2001, supra).

Pigs from the two PCV2 plasmid DNA-transfected groups (intrahepatic andintralymphoid) and the PCV2 virus-infected group (intranasal) hadsimilar lesions in brain, lung, heart, kidney, lymphoid tissues (tonsil,lymph nodes, spleen), ileum, and liver (see Table 4, below). Brainlesions were observed in 23/30 of the pigs from the three inoculatedgroups and were characterized as mild-to-moderate multifocallymphoplasmacytic meningoencephalitis with perivascular cuffing andgliosis. Lung lesions were observed in 28/30 PCV2-inoculated pigs andcharacterized as mild-to-moderate peribronchiolar lympho-plasmacytic andhistiocytic bronchointerstitial pneumonia (FIG. 3C). One pig from thePCV2 virus-infected Group 2 necropsied at 21 DPI, and one pig each fromthe two PCV2 plasmid DNA-transfected groups necropsied at 35 DPI hadulcerative and proliferative bronchiolitis with fibroplasia andgranulomatous inflammation in the lamina propria and peribronchiolarregions of bronchi. Mild multifocal lymphoplasmacytic myocarditis wasalso observed in 18/30 PCV2-inoculated pigs. In 14/30 of thePCV2-inoculated pigs, mild-to-moderate multifocal lymphoplasmacyticinterstitial nephritis was observed. No lesions were observed in thethymuses. Mild-to-moderate lymphoid depletion (FIG. 4B) and histiocyticreplacement of follicles was observed in the tonsil of 8/30, in thespleen of 7/30, and in the lymph nodes of 26/30 of the PCV2-inoculatedpigs. Moderate granulomatous lymphadenitis with giant cells (FIG. 4C)was observed at 21 DPI in three pigs inoculated intranasally with PCV2virus, and in one pig at 35 DPI in each of the PCV2 plasmidDNA-transfected groups. Mild lymphoplasmacytic and histiocyticenterocolitis were observed in 3/5 pigs in the PCV2 virus-infectedgroup, in 3/5 pigs in the PCV2 plasmid DNA intrahepatically-transfectedgroup, and 1/5 pigs in the PCV2 plasmid DNA intralymphoid-transfectedgroup at 35 DPI. One pig in each of the PCV2 plasmid DNA-transfectedgroups had mild lymphoid depletion with histiocytic replacement and lownumbers of giant cells in the Peyer's patches. Mild-to-moderatelymphoplasmacytic hepatitis was observed in 29/30 of the threePCV2-inoculated pigs. Low numbers of widely scattered individuallynecrotic hepatocytes surrounded by lymphohistiocytic inflammation wasobserved in one pig in each of the PCV2 plasmid DNA-transfected groupsat 21 DPI. Lesions in other tissues were unremarkable.

Microscopic lesions in the lung, liver and lymph nodes were scoredaccording to published scoring systems (Table 4, below) (P. G. Halbur etal., 2001, supra; P. G. Halbur et al., 1995, supra). There were noacceptable scoring systems for other tissues and organs. The averagescores of lesions in lung and lymph nodes in pigs of the threePCV2-inoculated groups were statistically different from those in thecontrol pigs of Group 1. The average scores of the liver lesions in pigsof the three PCV2-inoculated groups are not statistically different fromthose of control pigs.

TABLE 3 Gross Lesions of Lung and Lymph Nodes in Control andPCV2-Inoculated Pigs 21 DPI 35 DPI Route of Lymph Lymph Group InoculaInoculation Nodes Lung Nodes Lung 1 None 0/5^(a) 0/5 0/5 0/5 2 PCV2 livevirus^(b) Intranasal 5/5 1/5(0-1)^(c) 5/5 4/5(0-5) 3 PCV2 DNA^(d)Intrahepatic 2/5 2/5(0-2) 5/5 2/5(0-13) 4 PCV2 DNA^(d) Intralymphoid 4/55/5(0-1) 3/5 1/5(0-9) ^(a)Five pigs from each group were necropsied at21 DPI, and the remaining 5 pigs were necropsied at 35 DPI. Numberpositive/number tested. ^(b)A biologically pure and homogeneous PCV2virus stock generated by transfection of PK-15 cells with PCV2 molecularDNA clone. ^(c)Number with lesions/number tested (range of the estimatedpercent of the lung affected by grossly visible pneumonia lesions,0-100%) ^(d)Cloned PCV2 genomic DNA in pSK plasmid.

TABLE 4 Distribution of Histopathological Lesions in Control andPCV2-Inoculated Pigs Route of Group Inocula Inoculation DPI^(a) Lung^(b) Liver^(c) LN^(d) Spleen Thymus Ileum Brain Heart Kidney Tonsil 1None 21 0/5(0.0) 4/5(0.8) 0/5(0.0) 0/5 0/5 0/5 0/5 0/5 0/5 0/5 350/5(0.0) 4/5(0.8) 0/5(0.0) 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2 PCV2 virusIntranasal 21 5/5(1.6) 5/5(1.2) 3/5(1.2) 1/5 0/5 0/5 4/5 3/5 1/5 0/5 353/5(0.6) 4/5(1.0) 4/5(0.8) 3/5 0/5 3/5 4/5 0/5 1/5 3/5 3 PCV2 DNAIntrahepatic 21 5/5(1.0) 5/5(1.0) 5/5(1.0) 1/5 0/5 0/5 5/5 4/5 1/5 0/535 5/5(1.2) 5/5(1.0) 4/5(1.0) 2/5 0/5 3/5 3/5 4/5 5/5 3/5 4 PCV2 DNAIntralymphoid 21 5/5(1.2) 5/5(1.0) 5/5(0.8) 0/5 0/5 0/5 4/5 4/5 3/5 0/535 5/5(1.0) 5/5(1.2) 5/5(1.4) 0/5 0/5 1/5 3/5 3/5 3/5 2/5 ^(a)Dayspostinoculation (DPI): 5 animals from each group were necropsied at 21DPI and the remaining 5 animals from each group were necropsied at 35DPI ^(b) Number positive/number tested (Average histological lung score:0 = normal, 1 = mild interstitial pneumonia, 2 = moderate, 3 = severe)^(c)Number positive/number tested (Average histological liver score: 0 =normal, 1 = mild hepatitis, 2 = moderate, 3 = severe) ^(d)Numberpositive/number tested (Average histological lymphoid (LN) depletionscore: 0 = normal, 1 = mild, 2 = moderate, 3 = severe)

Example 8 Immunohistochemistry

Immunohistochemistry (IHC) detection of PCV2-specific antigen wasperformed on all tissues collected during necropsies at DPIs 21 and 35.A rabbit polyclonal PCV2-specific antiserum was used for the IHC, andthe general procedures have been previously described (S. D. Sorden etal., 1999, supra).

For the detection and tissue distribution of PCV2 antigen, IHC stainingof PCV2 antigen was done on brain, lungs, turbinate, heart, kidneys,tonsil, lymph nodes, spleen, thymus, ileum, liver, gall bladder andpancreas of all pigs necropsied at 21 and 35 DPI. All tissues from thecontrol pigs were negative for PCV2 antigen. Tissue distribution of PCV2antigen in the three PCV2-inoculated groups was similar (see Table 5,below). In the brain, the PCV2 antigen was found predominately inmononuclear cells, fibroblast-like cells, and endothelial cells in themeninges and choroid plexus and less often in endothelial cells andperivascular mononuclear cells in the cerebrum and cerebellum. In thelungs, PCV2 antigen was detected within alveolar and septal macrophagesand in fibroblast-like cells in the lamina propria of airways (FIG. 3D).In the heart, PCV2 antigen was detected in widely scattered macrophagesand endothelial cells. In kidneys, PCV2 antigen was detected withintubular epithelial cells and mononuclear cells in the interstitium. Inthe lymphoid tissues (lymph nodes, spleen, tonsil, and Peyer's patches),PCV2 antigen was detected primarily within macrophages anddendritic-like cells and giant cells within follicles (FIG. 4D). PCV2antigen was also detected within macrophages in the lamina propria ofthe small intestine. In the liver, PCV2 antigen was detected withinmononuclear cells and Kupffer cells. PCV2 antigen was not detected inturbinate, thymus, or gall bladder.

TABLE 5 Detection and Distribution of PCV2-Specific Antigen byImmunohistochemistry in Control and PCV2-Inoculated Pigs Route of GroupInocula Inoculation DPI^(a) Lung Liver LN Spleen Thymus Ileum BrainHeart Kidney Tonsil 1 None 21 0/5^(b) 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/50/5 35 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2 PCV2 virus Intranasal21 4/5 5/5 5/5 3/5 0/5 3/5 3/5 1/5 1/5 2/5 35 1/5 2/5 3/5 2/5 0/5 0/52/5 0/5 0/5 0/5 3 PCV2 DNA Intrahepatic 21 5/5 5/5 5/5 5/5 0/5 0/5 5/51/5 0/5 2/5 35 4/5 4/5 3/5 4/5 0/5 3/5 4/5 2/5 2/5 3/5 4 PCV2 DNAIntralymphoid 21 4/5 4/5 5/5 4/5 0/5 3/5 3/5 0/5 0/5 3/5 35 3/5 4/5 5/54/5 0/5 2/5 3/5 1/5 0/5 4/5 ^(a)Days postinoculation (DPI): 5 animalsfrom each group were necropsied at 21 DPI and the remaining 5 animalsfrom each group were necropsied at 35 DPI ^(b)Number positive/numbertested

Example 9 Construction of the Nonpathogenic PCV1 Infectious DNA Clone

The procedure used to construct a PCV1 infectious DNA clone isessentially the same as that described herein for PCV2. A pair of PCRprimers, KPNPCV1.0 set forth in SEQ ID NO:7 and KPNPCV1.L set forth inSEQ ID NO:8 (see Table 6, below), was designed based on the publishedsequence of PCV1. This pair of primers amplifies the complete genome ofPCV1 with an overlapping region containing the unique KpnI restrictionenzyme site. The DNA of the PCV1 virus was extracted from thecontaminated ATCC PK-15 cell line that was obtained from the AmericanType Culture Collection (ATCC accession number CCL-33). The PCV1 DNA wasextracted from the ATCC PK-15 cells persistently infected with PCV1,using the QIAmp DNA minikit (Qiagen, Inc., Valencia, Calif.). Theextracted DNA was amplified by PCR with AmpliTaq Gold Polymerase(Perkin-Elmer, Norwalk, Conn.). The PCR cycles consisted of an initialstep of 95° C. for 10 min., followed by 35 cycles of denaturation at 94°C. for 1 min., annealing at 48° C. for 1 min., extension at 72° C. for 2min., and a final extension at 72° C. for 7 min. The PCR product ofexpected size was separated by gel electrophoresis and purified by theglassmilk procedure using a Geneclean Kit (Bio 101, Inc., La Jolla,Calif.). The purified PCR product containing the complete PCV1 genomewas first ligated into the advanTAge plasmid vector (Clontech, PaloAlto, Calif.). Escherichia coli DH5a competent cells were used fortransformation. The recombinant plasmids were verified by restrictionenzyme digestion. The full length PCV1 genomic DNA was excised from theadvanTAge vector by digestion with KpnI restriction enzyme. Thefull-length PCV1 genomic DNA was ligated into pBluescript SK(+) (pSK)vector (Stratagene, La Jolla, Calif.) with T4 DNA ligase at 37° C.overnight. Recombinant plasmids containing the full-length PCV1 genomewere isolated with a Qiagen plasmid mini kit (Qiagen, Valencia, Calif.)and were verified by restriction enzyme digestion and DNA sequencing.The full-length PCV1 genomic DNA was excised from the pSK vector by KpnIdigestion, and dimmerized to make the PCV1 infectious DNA clone asdescribed above in Example 2 for the PCV2 infectious clone. These tandemdimers were made because the dimmerized tandem DNA clones areadvantageously found to be more efficient to transfect cells and produceinfectious virions. To make the tandem dimer of the PCV1 DNA, thedigested PCV1 genomic DNA was ligated with T4 DNA ligase at 37° C. foronly 10 min., which favors the production of tandem dimers. The tandemdimers were subsequently cloned into pBluescript SK(+) (pSK) vector(Stratagene, La Jolla, Calif.). Recombinant plasmids containing tandemdimers of PCV1 genome (herein referred to as “PCV1 DNA clone”) wereconfirmed by PCR, restriction enzyme digestion, and DNA sequencing. TheDNA concentration of the recombinant plasmids was determinedspectrophotometrically.

TABLE 6 Oligonucleotide Primers Employed in this Invention PrimerDirection Primer Sequence Application Construction  primers: KPNPCV1.U.>^(a) 5′-TTTGGTACCCGAAGGCCGATT-′3  PCV1 DNA  (corresponds to SEQ ID NO: 7) clone construction KPNPCV1.L. <5′-ATTGGTACCTCCGTGGATTGTTCT-′3  PCV1 DNA   (corresponds to SEQ ID NO: 8)clone construction Hpa I-2 <5′-GAAGTTAACCCTAAATGAATAAAAATAAAAACCATTACG-′3 PCV1-2 DNA  (corresponds to SEQ ID NO: 9) clone construction Nar I-3 >5′-GGTGGCGCCTCCTTGGATACGTCATCCTATAAAAGTG-′3 PCV1-2 DNA  (corresponds to SEQ ID NO: 10) clone construction Psi I-5 >5′-AGGTTATAAGTGGGGGGTCTTTAAGATTAA-′3 PCV1-2 DNA  (corresponds to SEQ ID NO: 11) clone construction Acl I-6 <5′-GGAAACGTTACCGCAGAAGAAGACACC-′3 PCV1-2 DNA  (corresponds to SEQ ID NO: 12) clone construction Bgl-II-ORF2 >5′-ACTATAGATCTTTATTCATTTAGAGGGTCTTTCAG-′3 PCV2-1 DNA  (corresponds to SEQ ID NO: 13) clone construction SpH-I-ORF2 <5′-TACGGGCATGCATGACGTGGCCAAGGAGG-′3 PCV2-1 DNA  (corresponds to SEQ ID NO: 14) clone construction Bgl-II-PCV2 <5′-AGACGAGATCTATGAATAATAAAAACCATTACGAAG-′3 PCV2-1 DNA  (corresponds to SEQ ID NO: 15) clone construction SpH-I-PCV2 >5′-CGTAAGCATGCAGCTGAAAACGAAAGAAGTG-′3 PCV2-1 DNA  (corresponds to SEQ ID NO: 16) clone construction Detection  primers:MCV1 > 5′-GCTGAACTTTTGAAAGTGAGCGGG-′3  PCV1 and PCV2 (corresponds to SEQ ID NO 17) detection MCV2 <5′-TCACACAGTCTCAGTAGATCATCCCA-′3  PCV1 and PCV2 (corresponds to SEQ ID NO: 18) detection Orf.PCV1 <5′-CCAACTTTGTAACCCCCTCCA-′3  PCV1 and   (corresponds to SEQ ID NO: 19)PCV2-1 detection Gen.PCV1 > 5′-GTGGACCCACCCTGTGCC-′3  PCV1 and  (corresponds to SEQ ID NO: 20) PCV1-2 detection Nested.OrfPCV1 <5′-CCAGCTGTGGCTCCATTTAA-′3  PCV1 and   (corresponds to SEQ ID NO: 21)PCV2-1 detection Nested.Gen.PCV1 > 5′-TTCCCATATAAAATAAATTACTGAGTCTT-′3PCV1 and   (corresponds to SEQ ID NO: 22) PCV1-2 detection Orf.PCV2 <5′-CAGTCAGAACGCCCTCCTG-′3  PCV2 and   (corresponds to SEQ ID NO: 23)PCV1-2 detection Gen.PCV2 > 5′-CCTAGAAACAAGTGGTGGGATG-′3  PCV2 and  (corresponds to SEQ ID NO: 24) PCV2-1 detection Nested.OrfPCV2 <5′-TTGTAACAAAGGCCACAGC-′3  PCV2 and   (corresponds to SEQ ID NO: 25)PCV1-2 detection Nested.Gen.PCV2 > 5′-GTGTGATCGATATCCATTGACTG-′3 PCV2 and   (corresponds to SEQ ID NO: 26) PCV2-1 detection ^(a)Primerdirection.

Example 10 Evaluation of Infectivity of the PCV1 DNA Clone whenTransfected into PK-15 Cells Free of Virus Contamination

The infectivity of the PCV1 molecular DNA clone was determined by invitro transfection of the PK-15 cells. IFA with PCV1 specific monoclonalantibody (a gift from Dr. Gordon Allan, Belfast, U.K.) confirmed thatthe PCV1 molecular DNA clone is infectious in PK-15 cells. PCV1-specificantigen was visualized by IFA in the nucleus, and to a lesser degreecytoplasm of the transfected cells. The cells mock-transfected with theempty pSK vector remained negative for PCV1 antigen.

Example 11 Construction of a Chimeric PCV1-2 Viral DNA Clone

A chimeric virus was constructed between the nonpathogenic PCV1 and thePMWS-associated PCV2 by using infectious DNA clones of PCV1 and PCV2. Toconstruct a chimeric PCV1-2 DNA clone, the ORF2 capsid gene of thenonpathogenic PCV1 was removed from the PCV1 infectious DNA clone, andreplaced with the immunogenic ORF2 capsid gene of the pathogenic PCV2 inthe genome backbone of PCV1 (see FIGS. 5 and 6). Two pairs of PCRprimers were designed. The first primer pair for PCV2 ORF2, Psi I-5 setforth in SEQ ID NO:11 and Ad 1-6 set forth in SEQ ID NO:12, was designedwith point mutations at the 5′ ends of the primers to create restrictionenzyme sites AclI and PsiI to amplify the ORF2 gene of PCV2 andintroduce flanking PsiI and AclI restriction enzyme sites by pointmutation. The PCR reaction for the PCV2 ORF2 amplification consisted ofan initial step at 95° C. for 9 min., followed by 38 cycles ofdenaturation at 95° C. for 1 min., annealing at 48° C. for 1 min.,extension at 72° C. for 1 min., and a final extension at 72° C. for 7min.

A second pair of PCR primers, Hpa 1-2 set forth in SEQ ID NO:9 and Nar1-3 set forth in SEQ ID NO:10, was designed for the amplification of thepSK+ vector and its PCV1 genome insert. Point mutations were introducedat the 5′ ends of the PCR primers to create flanking restriction enzymesites NarI and HpaI. This primer pair amplified the pSK+ vector and itsinsert PCV1 genomic DNA lacking the ORF2 capsid gene, that is, the PCV1genome minus the PCV1 ORF2 (pSK-PCV1 ΔORF2) by using the PCV1 infectiousDNA clone as the PCR template. The PCR reaction consisted of an initialstep at 95° C. for 9 min., followed by 38 cycles of denaturation at 95°C. for 1 min., annealing at 50° C. for 1 min., extension at 72° C. for3.5 min., and a final extension at 72° C. for 7 min. The PCV2 ORF2 PCRproduct was digested with the AclI and PsiI to remove the introducedpoint mutations. The pSK-PCV1 ΔORF2 product (the pSK vector-PCV1 genomePCR product lacking ORF2 gene of PCV1) was digested with the NarI andHpaI to remove the PCR introduced point mutations. The latter digestionproduced a sticky end and a blunt end complementary to the PCV2 ORF2 PCRproduct digested by the AclI and PsiI restriction enzymes. The digestedPCV2 ORF2 product and the ORF2-deleted pSK-PCV1 product were ligatedwith T4 DNA ligase to form the chimeric PCV1-2 genomic DNA clone, inwhich the ORF2 gene of PCV1 is replaced with the ORF2 gene of PCV2. Oncethe two PCR products were digested and religated, all the PCR introducedpoint mutations used to facilitate cloning were removed in the resultingchimeric clone. Escherichia coli DH5a competent cells were transformed.The recombinant plasmids containing the chimeric DNA clone were isolatedand confirmed by PCR, restriction enzyme digestion and partial DNAsequencing. The full-length chimeric PCV1-2 genome was excised from thepSK+ vector (the recombinant plasmid) with KpnI digestion. The chimericDNA genome was then dimmerized by a short 10-minute ligation reactionwith T4 DNA ligase that favors the formation of linear dimers to producethe PCV1-2 chimeric infectious DNA clone (FIG. 6). The recombinantplasmids containing two copies of the chimeric viral genome wereconfirmed by PCR, restriction enzyme digestion and DNA sequencing.

Example 12 Evaluation of In Vitro Infectivity of PCV1-2 Chimeric DNAClone

The viability of the chimeric PCV DNA clone (nonpathogenic PCV1 with theimmunogenic capsid gene of PCV2) was tested in PK-15 cells. When PK-15cells were transfected with the chimeric viral DNA clone, viral antigenspecific for PCV2 ORF2 capsid was detected by IFA at about 2 dayspost-transfection. The PCV1 capsid antigen was not detected intransfected cells. This experiment indicated that the chimeric DNA cloneis infectious in vitro, is replicating in PK-15 cells and producing theimmunogenic capsid protein of PCV2.

Example 13 Construction of a Reciprocal Chimeric PCV2-1 DNA Clone

To construct a reciprocal PCV2-1 chimeric DNA clone, the ORF2 capsidgene of PCV2 is replaced by that of the non-pathogenic PCV1 in thegenome backbone of the pathogenic PCV2 (FIG. 6). Two PCR primer pairswere designed: the pair, Bgl-II-ORF2 set forth in SEQ ID NO:13 andSpH-I-ORF2 set forth in SEQ ID NO:14, amplifies the PCV1 ORF2 gene andintroduces flanking BglII and SpHI restriction enzyme sites by pointmutation. The second PCR primer pair, Bgl-II-PCV2 set forth in SEQ IDNO:15 and SpH-I-PCV2 set forth in SEQ ID NO:16, amplified the pSK vectorand the PCV2 genome minus the ORF2 gene (pSK-PCV2 ΔORF2) by using thePCV2 infectious DNA clone as the PCR template, and introduced flankingrestriction enzymes sites BglII and Spill by point mutation. ThepSK-PCV2 ΔORF2 product and the PCV1 ORF2 PCR product were digested byBglII and SpHI restriction enzymes to produce complementary sticky andblunt ends ligated together. After transformation into E. Coli cells,the authentic recombinant plasmids were isolated and confirmed by enzymedigestion and partial DNA sequencing. The full-length reciprocalchimeric PCV2-1 genome was excised from the recombinant plasmid by SacIIdigestion, and dimmerized as described herein to produce the reciprocalchimeric PCV2-1 infectious clone.

Example 14 In Vitro Transfection of PK-15 Cells with PCV1, PCV2, PCV1-2and PCV2-1 DNA Clones

The infectivity of PCV2 clone in vitro and in vivo has been demonstratedin the above Examples 3-5. To test the infectivity of the PCV1 and twochimeric clones in vitro, PK-15 cells free of PCV1 contaminationprepared per the method of Example 1 were grown in 8-well LabTekchambers slides (Nalge Nunc Int., Denmark). When the PK-15 cells reachedabout 80% confluency, cells were transfected with PCV1, PCV2, PCV1-2 andPCV2-1 DNA clones respectively, using the Lipofectamine Plus Reagentaccording to the protocols supplied by the manufacturer (LifeTechnologies, Inc.). Mock-transfected cells with empty pSK vector wereincluded as controls. Three days after transfection, the cells werefixed with a solution containing 80% acetone and 20% methanol at 4° C.for 20 min. Evidence of infectivity and virus replication in cellstransfected with the PCV1 and PCV2-1 DNA clones were confirmed byindirect immunofluorescence assay (IFA) using monoclonal antibodyagainst PCV1 ORF2 capsid gene, kindly provided by Dr. G. M. Allan (G. M.Allan et al., “Production, preliminary characterization and applicationsof monoclonal antibodies to porcine circovirus,” Vet. Immunol.Immunopathol. 43:357-371 (1994)). The fixed cells were washed withphosphate buffered saline (PBS) and incubated with 1:20 diluted PCV1monoclonal antibody at 37° C. for 1 hour. The cells were then washedthree times with PBS buffer and incubated with fluoresceinisothiocyanate (FITC) labeled goat anti-mouse immunoglobulin G(Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.) at 37° C. for45 min. After washing three times with PBS buffer, the slides weremounted with fluoromount-G, coverslipped, and examined under afluorescence microscope. The infectivity of cells transfected with thePCV2 and the chimeric PCV1-2 DNA clones were confirmed by IFA usingantibody specific for PCV2, as previously described in Example 4.

The infectivity of the PCV1, the chimeric PCV1-2 DNA and the reciprocalchimeric PCV2-1 DNA clones were substantiated by the in vitrotransfection of PK-15 cells. Two identical copies of the complete PCV1genome were ligated in tandem into the pSK vector to produce the PCV1DNA clone (FIG. 6). The chimeric PCV1-2 DNA clone had the ORF2 capsidgene of PCV1 replaced by that of the pathogenic PCV2 in the backbone ofthe nonpathogenic PCV1 genome. The reciprocal chimeric PCV2-1 DNA clonehad the ORF2 capsid gene of PCV2 replaced by that of the nonpathogenicPCV1 in the backbone of the pathogenic PCV2 genome. If infectious invitro, the chimeric PCV1-2 DNA clone will produce the ORF2 capsidantigen of PCV2 and the reciprocal chimeric PCV2-1 DNA clone willexpress PCV1 ORF2 capsid antigen in transfected PK-15 cells. The resultsshowed that the PCV1, the chimeric PCV1-2 and the reciprocal chimericPCV2-1 DNA clones were all surprisingly shown to be infectious whentransfected into PK-15 cells and expressed respective viral capsidantigen proteins as demonstrated by IFA using antibodies specific forPCV1 or PCV2. IFA using monoclonal antibodies against PCV1 ORF2 andantibodies against PCV2 confirmed that the PCV1 DNA and the PCV1-2 DNAclones were infectious. IFA using PCV1 ORF2-specific monoclonal antibodyshowed that the PCV1-2 chimeric DNA clone was also infectious. About10-20% of the transfected PK-15 cells were positive for PCV1 capsidantigen and PCV2 antigen, and expressed PCV1 ORF2 antigen, within thenucleus of transfected cells (FIG. 7).

Example 15 Experimental Inoculation of Pigs with PCV1, PCV2, ChimericPCV1-2 and Reciprocal Chimeric PCV2-1 DNA Clones

To evaluate the immunogenicity and pathogenicity of the chimeric DNAclones, forty specific-pathogen-free (SPF) pigs of 4-6 weeks of age wererandomly assigned into five rooms of 8 animals each. Prior toinoculation, animals were tested for antibodies to PCV, PRRSV, PPV, andswine hepatitis E virus. In addition, pre-inoculation serum samples weretested by PCR for PCV1 and PCV2 nucleic acid to confirm that the pigsare not naturally infected by either of the viruses. The PCV1, PCV2,PCV1-2 and PCV2-1 DNA clones were all inoculated by direct injection ofthe cloned plasmid DNA into the superficial iliac lymph nodes of pigs.Pigs in Group 1 received phosphate buffered saline (PBS buffer) andserved as the negative control. Group 2 pigs were each injected into thesuperficial iliac lymph nodes with 200 μg of infectious PCV1 DNA clone.Group 3 pigs were each injected with 200 μg of infectious PCV2 DNAclone. Group 4 pigs each received injections of 200 μg of infectiouschimeric PCV1-2 DNA clone. Group 5 pigs each received 200 μg of theinfectious reciprocal chimeric PCV2-1 DNA clone. All animals weremonitored daily for clinical signs of disease. Serum samples werecollected from each animal at −2, 7, 14, 21, 28, 35, 42 and 49 dayspost-inoculation (DPI). At 21 DPI, four randomly selected animals fromeach group were necropsied. The remaining four animals in each groupwere necropsied at 49 DPI. Various tissues and organs were collectedduring necropsy as previously described in Example 7, and processed forhistological examination.

The immunogenicity of the PCV1, the PCV2 and the chimeric infectious DNAclones was examined in the pigs. Serum samples collected from allcontrol and inoculated animals at −2 (0), 7, 14, 21, 28, 35, 42 and 49DPIs were assayed for PCV1, PCV2, PCV1-2 and PCV2-1 viremia by PCRdetection of clone-specific DNA sequences, for anti-PCV1 antibody by IFAand for anti-PCV2 ORF2 antibody by ELISA. Prior to inoculation at −2DPI, animals from all five groups tested negative by PCR for both PCV1and PCV2 DNA.

Negative control animals were negative for both PCV1 and PCV2 viremiathroughout the study (see Table 7, below). Five pigs in the uninoculatedcontrol group had detectable PCV2 maternal antibody at −2 DPI and 2 pigshad detectable PCV1 maternal antibodies at 7 DPI (see Table 8, below).The maternal antibodies to both PCV1 and PCV2 in these piglets waned by21 DPI. No seroconversion to either PCV1 or PCV2 was detected in any ofthe 8 uninoculated control pigs throughout the study.

In the PCV1 inoculated group, viremia was first detected in aninoculated pig at 7 DPI (Table 7, below), and was last detected at 35DPI. Five out of 8 animals inoculated with PCV1 infectious DNA clonewere positive for PCV1 viremia. Average length of continuous PCV1viremia was 0.625 weeks. By 21 DPI, all animals in the PCV1 inoculatedgroup had seroconverted to PCV1 and remained positive to PCV1 antibodiesuntil the end of the study at 49 DPI.

The PCV2 DNA clone is shown herein to be infectious in pigs. In the PCV2DNA clone inoculated group, PCV2 viremia was first detected at 7 DPI(Table 7, below). By 21 DPI, all PCV2 inoculated Group 3 animals werepositive for PCV2 viremia. The average length of PCV2 viremia was 2.12weeks. Two pigs in the PCV2 inoculated group had detectable levels ofmaternal PCV2 antibodies at 7 DPI (Table 8, below), and the maternalantibodies in these piglets waned by 14 DPI. Seroconversion to PCV2,assayed by a PCV2-specific ELISA, was first detected at 35 DPI. By 42DPI, all pigs inoculated with PCV2 infectious DNA clone hadseroconverted to PCV2.

In Group 4 pigs inoculated with PCV1-2 chimeric infectious DNA clone,viremia specific for the chimeric virus was first detected at 14 DPI(Table 7, below). Four out of 7 inoculated animals became viremic toPCV1-2 between 14 DPI and 42 DPI. The average length of chimeric PCV1-2viremia was 1 week. One pig had detectable levels of maternal PCV2antibodies at 7 and 14 DPI, but the maternal antibody waned by 21 DPI(Table 8, below). Seroconversion to PCV2 ORF2-specific antibody firstoccurred at 28 DPI. By 49 DPI, all pigs inoculated with chimeric PCV1-2DNA clone had seroconverted to PCV2 ORF2-specific antibody.

In pigs inoculated with the reciprocal chimeric PCV2-1 clone, viral DNAspecific for PCV2-1 chimeric virus was not detected in serum samples(Table 7, below). However, by 21 DPI all animals in Group 5seroconverted to PCV1 antibody. PCR products amplified from selectedpigs in each group were sequenced and confirmed to be the authenticrespective infectious clones used in the inoculation in each group.

TABLE 7 Detection of Viremia by Nested PCR in Sera of Inoculated andControl Pigs DPI Group Inoculum −2 7 14 21 28 35 42 49 Total 1 PBS^(a)0/8^(b) 0/8 0/8 0/8 0/4 0/4 0/4 0/4 0/8 2 PCV1 DNA^(c) 0/8 1/8 1/8 2/80/4 2/4 0/4 0/4 5/8 3 PCV2 DNA^(c) 0/8 3/8 6/8 7/8 1/4 2/4 2/4 0/4 8/8 4PCV1-2 DNA^(c) 0/7 0/7 1/7 2/7 2/4 2/4 2/4 0/4 4/7 5 PCV2-1 DNA^(c) 0/80/8 0/8 0/8 0/4 0/4 0/4 0/4 0/8 ^(a)Phosphate buffered saline (PBS) usedas negative control ^(b)Eight pigs in each group; number positive/numbertested ^(c)Cloned genomic PCV or chimeric PCV DNA in pSK plasmid

TABLE 8 Seroconversion to Antibodies Against PCV2 in Pigs Inoculatedwith PCV2 or Chimeric PCV1-2 DNA Clones and Seroconversion to AntibodiesAgainst PCV1 in Pigs Inoculated with PCV1 or PCV2-1 DNA Clones DPI^(c)Group Inoculum^(a) Antibody Tested For^(b) −2 7 14 21 28 35 42 49 1 PBSPCV1 NA 2/8 2/8 1/8 0/4 0/4 0/4 0/4 PCV2 5/8 5/8 2/8 0/8 0/4 0/4 0/4 0/42 PCV1 DNA PCV1 ORF2 NA 3/8 2/8 8/8 4/4 4/4 4/4 4/4 3 PCV2 DNA PCV2complete virus 3/8 2/8 0/8 0/8 0/4 3/4 4/4 4/4 4 PCV1-2 DNA PCV2complete virus 2/8 1/8 1/8 0/8 1/4 1/4 3/4 4/4 5 PCV2-1 DNA PCV1 ORF2 NA3/8 3/8 8/8 3/4 3/4 4/4 4/4 ^(a)Phosphate buffered saline (PBS) used asnegative control. The inocula were cloned genomic PCV and PCV chimericDNA in pSK plasmid ^(b)PCV1 antibody to ORF2 was measured with anindirect immunofluorescence assay specific for PCV1 antigen. PCV2antibody was measured with an enzyme-linked immunosorbent assay.^(c)Days post inoculation. number positive/number tested.

Example 16 Clinical Evaluation

Pigs were weighed on 0 DPI and at the time of necropsies. Rectaltemperatures and clinical respiratory scores, ranging from 0 to 6(0=normal; 6=severe) (P. G. Halbur et al., “Comparison of thepathogenicity of two U.S. porcine reproductive and respiratory syndromevirus isolates with that of the Lelystad virus,” Vet. Pathol. 32:648-660(1995)), were recorded every other day from 0 to 49 DPI. Clinicalobservations, including evidence of central nervous system disease,liver disease (icterus), muscoloskeletal disease and changes in bodycondition, were also recorded daily. A team of two people performed allclinical evaluations.

None of the control or inoculated pigs showed obvious signs of thefull-spectrum clinical PMWS. There were no differences in weight gain ormean rectal temperatures between any of the groups. One of the pigs fromPCV1-2 inoculated Group 3 died one day after inoculation. After necropsyand clinical analysis, no pathogenic agent was detected and death wasnot associated with the inoculation procedure or the chimeric PCV1-2virus.

Example 17 Gross Pathology and Histopathology

Four pigs from each group were necropsied at 21 and 49 DPI,respectively. The necropsy team was blinded to infection status of thepigs at necropsy. Complete necropsies were performed on all pigs. Anestimated percentage of the lung with grossly visible pneumonia wasrecorded for each pig based on a previously described scoring system (P.G. Halbur et al., 1995, supra). Other lesions such as enlargement oflymph nodes were noted separately. Sections for histopathologicexamination were taken from nasal turbinate, lungs (seven sections)(id.), heart, brain, lymph nodes (tracheobronchial, iliac, mesenteric,subinguinal), tonsil, thymus, liver, gall bladder, spleen, joints, smallintestine, colon, pancreas, and kidney. The tissues were examined in ablinded fashion and given a subjective score for severity of lung, lymphnode, and liver lesions as described in Example 7. Lung scores rangedfrom 0 (normal) to 3 (severe lymphohistiocytic interstitial pneumonia).Liver scores ranged from 0 (normal) to 3 (severe lymphohistiocytichepatitis). Lymph node scores were for an estimated amount of lymphoiddepletion of follicles ranging from 0 (normal or no lymphoid depletion)to 3 (severe lymphoid depletion and histiocytic replacement offollicles).

To determine the pathogenicity of PCV1, PCV2, chimeric PCV1-2 andreciprocal chimeric PCV2-1 infectious DNA clones in pigs, gross lesionswere examined first. The results are shown in Table 9 below. Lymph nodesof animals from the uninoculated control Group 1 were normal at both 21and 49 DPIs. Pigs in the four inoculated groups had variable degrees ofgross lesions limited to the lymph nodes. In PCV1 inoculated Group 2pigs, lymph nodes were grossly normal at 21 DPI, however, mild tomoderate swelling and discoloration of lymph nodes was detected at 49DPI. All PCV2 inoculated Group 3 pigs had enlarged lymph nodes two tofive times the normal size, that were firm and tan colored at both 21and 49 DPIs. Lymph nodes from chimeric PCV1-2 inoculated animals weremild to moderately swollen and discolored at both 21 and 49 DPIs in 5out of 7 pigs. In Group 5 pigs, inoculated with the PCV2-1 clone, 1 outof 8 animals had mild swelling and discoloration of the lymph nodes at21 DPI. The average scores of gross lesions of the lymph nodes in pigsinoculated with chimeric PCV1-2 clone were not statistically differentfrom those in Groups 1, 2, and 5, but were statistically different fromthose of the pathogenic PCV2 inoculated Group 3 pigs at both 21 and 49DPIs. Average lymphoid gross lesion scores on 49 DPI from the PCV1,PCV2, and PCV1-2 inoculated animals were not statistically differentfrom each other, but were all statistically different from the averagegross lesion scores of Groups 1 and 5.

Next, microscopic lesions were examined. The results are shown in Table10 below. No microscopic lesions were detected in either uninoculatedcontrol Group 1 pigs or PCV1 inoculated Group 2 pigs at any DPI.Microscopic lung lesions characterized as mild peribronchiolarlymphoplasmacytic and histiocytic bronchointerstitial pneumonia, wereobserved in 1 out 8 of the PCV2 inoculated pigs. In PCV1-2 and PCV2-1inoculated animals, no microscopic lesions were observed in the lungs.No lesions were observed in the thymuses of any inoculated pigs. Mildmultifocal lymphoplasmacytic myocarditis was observed in 2 of 8 pigs inthe PCV2 inoculated group. Heart tissues from PCV1-2 and PCV2-1inoculated animals were free of microscopic lesions. Mild multifocallymphoplasmacytic interstitial nephritis was observed in 4 out of 8 pigsin PCV2 inoculated group, in 2 out of 7 PCV1-2 inoculated pigs and in 1out of 8 PCV2-1 inoculated pigs. Mild-to-moderate lymphoid depletion andhistiocytic replacement of follicles were observed in the tonsil in 5out of 8 pigs, in the spleen in 3 out of 8 pigs, and in the lymph nodesin 8 out of 8 pigs in the PCV2-inoculated group. In the chimeric PCV1-2inoculated animals, mild lymphoid depletion and histiocytic replacementof follicles were observed in the lymph nodes of 2 out of 7 pigs butwere not detected in either the spleen or tonsils. No lymphoid depletionand histiocytic replacement of follicles were observed in the lymphnodes, spleen or tonsils of the reciprocal chimeric PCV2-1 inoculatedanimals. Mild-to-moderate lymphoplasmacytic hepatitis was observed in 7out of the 8 PCV2-inoculated pigs. Mild lymphoplasmacytic hepatitis wasobserved in 2 out of the 7 chimeric PCV1-2 inoculated pigs. Nolymphoplasmacytic hepatitis was observed in reciprocal chimeric PCV2-1inoculated pigs. Lesions in other tissues were unremarkable.

Microscopic lesions in the lung, liver, and lymph nodes were scoredaccording to a published scoring system (P. G. Halbur et al., 1995,supra). The results are shown in Table 10 below. Average scores oflesions in lymph nodes in pigs from the chimeric PCV1-2 inoculated Group4 were similar to those from Groups 1, 2 and 5 but were statisticallydifferent from those of the pathogenic PCV2 inoculated Group 3 pigs, atboth 21 and 49 DPIs. Average microscopic liver lesion scores from thechimeric PCV1-2 inoculated group at 21 DPI were statistically differentfrom those of PCV2 inoculated Group 3 animals but were similar to thoseof Groups 1, 2 and 5 pigs at 21 DPI. At 49 DPI, the average microscopicliver scores from Group 4 chimeric PCV1-2 inoculated pigs were notstatistically different from those of Groups 1, 2, 3 and 5 pigs. Therewere no acceptable scoring systems for other tissues or organs.

TABLE 9 Gross Lesions of Lymph Nodes in Control and Inoculated PigsDPI^(b) Group Inoculum^(a) 21 49 1 PBS 0/4(0.0)  0/4(0.0)  2 PCV1 DNA0/4(0.0)  4/4(1.5)  3 PCV2 DNA 4/4(2.5)  4/4(2.25) 4 PCV1-2 DNA2/3(0.66) 3/4(1.25) 5 PCV2-1 DNA 1/4(0.25) 0/4(0.0)  ^(a)Phosphatebuffered saline (PBS) used as negative control. The inocula were clonedgenomic PCV or chimeric PCV DNA in pSK plasmid. ^(b)Four pigs from eachgroup were necropsied at 21 DPI and the remaining pigs were necropsiedat 49 DPI; number positive/number tested. Number with lesions/numbertested (range of estimated severety lymph node enlargement)

TABLE 10 Distribution of Histopathological Lesions in DifferentTissues/Organs from Control and Inoculated Pigs Group Inoculum^(a)DPI^(b) Lung^(c) Liver^(d) Lymph Nodes^(e) Spleen Thymus Ileum BrainHeart Kidney Tonsil 1 PBS 21 0/4(0.0) 0/4(0.0) 0/4(0.0) 0/4 0/4 0/4 0/40/4 0/4 0/4 49 0/4(0.0) 0/4(0.0) 0/4(0.0) 0/4 0/4 0/4 0/4 0/4 0/4 0/4 2PCV1 DNA 21 0/4(0.0) 0/4(0.0) 0/4(0.0) 0/4 0/4 0/4 0/4 0/4 0/4 0/4 490/4(0.0) 0/4(0.0) 0/4(0.0) 0/4 0/4 0/4 0/4 0/4 0/4 0/4 3 PCV2 DNA 210/4(0.0) 4/4(1.5) 4/4(1.75) 3/4 0/4 0/4 1/4 1/4 2/4 3/4 49 1/4(0.25)3/4(0.75) 4/4(1.0) 0/4 0/4 0/4 0/4 1/4 2/4 2/4 4 PCV1-2 DNA 21 0/3(0.0)1/3(0.33) 1/3(0.33) 0/4 0/4 0/4 0/4 0/4 0/4 0/4 49 0/4(0.0) 1/4(0.25)1/4(0.25) 0/4 0/4 0/4 0/4 0/4 2/4 0/4 5 PCV2-1 DNA 21 0/4(0.0) 0/4(0.0)0/4 (0.0) 0/4 0/4 0/4 0/4 0/4 0/4 0/4 49 0/4(0.0) 0/4(0.0) 1/4(0.25) 0/40/4 0/4 0/4 0/4 0/4 0/4 ^(a)Phosphate buffered saline (PBS) used asnegative control. The inocula were cloned genomic PCV or chimeric PCVDNA in pSK plasmid. ^(b)Four pigs from each group were necropsied at 21DPI and the remaining pigs were necropsied at 49 DPI. ^(c)Numberpositive/number tested (average histological lung score: 0, normal; 1,mild interstitial pneumonia; 2, moderate; 3, severe). ^(d)Numberpositive/number tested (Average histiological liver sore: 0, normal; 1,mild hepatitis; 2, moderate; 3, severe.) ^(e)Number positive/numbertested (Average histiological lymphoid (lymph nodes) depletion score: 0,normal; 1, mild; 2, moderate; 3, severe.)

Example 18 Serology

Blood was collected from all pigs at −2, 7, 14, 21, 28, 35, 42 and 49DPIs. Serum antibodies to PRRSV were assayed using Herd Check PRRSVELISA (IDEXX Laboratories, Westbrook, Mass.). Serum antibodies to PPVwere detected by a hemagglutination inhibition (HI) assay (H. S. Joo etal., “A standardized haemagglutination inhibition test for porcineparvovirus antibody,” Aust. Vet. J. 52:422-424 (1976)). Serum antibodiesto PCV2 were detected by a modified indirect ELISA based on therecombinant ORF2 capsid protein of PCV2 as described hereinabove (seealso P. Nawagitgul et al., “Modified indirect porcine circovirus (PCV)type 2-based and recombinant capsid protein (ORF2)-based ELISA for thedetection of antibodies to PCV,” Immunol. Clin. Diagn. Lab Immunol.1:33-40 (2002)). Serum antibodies to PCV1 were detected by an indirectimmunofluorescence assay (IFA). PK-15 cells infected with PCV1 weregrown on eight-well LabTek chamber slides. When the infected PK-15 cellsreach about 95-100% confluency, the infected cells were fixed with asolution containing 80% acetone and 20% methanol at 4° C. for 20 min.The fixed cells were washed once with PBS buffer. One hundredmicroliters of 1:10 diluted pig serum sample in PBS was added to thechambers, and incubated for 1 hour at 37° C. The cells were then washedthree times with PBS and incubated for 45 min. at 37° C. withFITC-labeled goat anti-swine secondary antibody. The slides weresubsequently washed three times with PBS, mounted with fluoromount-G,coverslipped and examined under a fluorescent microscope. For thepositive control, PCV1 infected cells were incubated with a diluted PCV1specific monoclonal antibody (gift of Dr. G. M. Allan), followed by anincubation with FITC-labeled goat anti-mouse IgG (Kirkegaard & PerryLaboratories, Inc., Gaithersburg, Md.). For the negative control, PCV1infected cells were incubated with 1:10 diluted swine serum free of PCV1and PCV2 antibody, followed by an incubation with FITC-labeled goatanti-swine IgG (Kirkegaard & Perry Laboratories, Inc., Gaithersburg,Md.).

Example 19 PCR Detection

To detect PCV1, PCV2, chimeric PCV1-2 and reciprocal chimeric PCV2-1viremia in sera from inoculated pigs, serum samples collected atdifferent DPIs were tested by PCR. Viral DNA was extracted from 100 μlof each serum sample using DNAzol reagent according to themanufacturer's protocol (Molecular Research Center, Cincinnati, Ohio).The extracted DNA was resuspended in DNase, RNase and proteinase-freewater. To amplify clone-specific genomic sequences of PCV1, PCV2,chimeric PCV1-2 and chimeric reciprocal PCV2-1, two sets of nested PCRprimer pairs were designed (Table 6, above). The first set of nestedprimers was designed based on published PCV1 sequences. Primers Gen.PCV1set forth in SEQ ID NO:20 and Orf.PCV1 set forth in SEQ ID NO:19amplified a 400 bp fragment in the presence of the PCV1 genome. Thenested primers, nested.Gen.PCV1 set forth in SEQ ID NO:22 andnested.Orf.PCV1 set forth in SEQ ID NO:21, amplified a 220 bp fragment.

To detect PCV2 viremia, PCV2 primer pair Gen.PCV2 set forth in SEQ IDNO:24 and Orf.PCV2 set forth in SEQ ID NO:23 amplified a 900 bp fragmentin the presence of PCV2 in the first round of PCR. Primersnested.Gen.PCV2 set forth in SEQ ID NO:26 and nested.Orf.PCV2 set forthin SEQ ID NO:25 amplified a 600 bp fragment in the nested PCR.

To detect chimeric PCV1-2 viremia, the first round of PCR reactionemployed the PCV1-specific primer Gen.PCV1 set forth in SEQ ID NO:20 andthe PCV2 ORF2-specific primer Orf.PCV2 set forth in SEQ ID NO:23 toamplify a chimeric fragment of 580 bp. For the nested PCR, PCV1-specificprimer nested.Gen.PCV1 set forth in SEQ ID NO:22 and the PCV2ORF2-specific primer nested.Orf.PCV2 set forth in SEQ ID NO:25 were usedto amplify a chimeric fragment of 370 bp.

To detect reciprocal chimeric PCV2-1 viremia, the first round of PCRemployed the PCV2-specific primer Gen.PCV2 set forth in SEQ ID NO:24 andthe PCV1 ORF2-specific primer Orf.PCV1 set forth in SEQ ID NO:19 toamplify a chimeric fragment of 700 bp. For the nested PCR, thePCV2-specific primer nested.Gen.PCV2 set forth in SEQ ID NO:26 and thePCV1 ORF2-specific primer nested.Orf.PCV1 set forth in SEQ ID NO:21 wereused to amplify a 460 bp chimeric fragment. All PCR parameters wereessentially the same, consisting of 38 cycles of denaturation at 94° C.for 1 min., annealing at 45° C. for 1 min., and extension at 72° C. for1.5 min. The serum samples from negative control pigs were tested by aPCR-RFLP diagnostic assay, which can detect and differentiate both PCV1and PCV2 as described previously (M. Fenaux et al., “Geneticcharacterization of type 2 porcine circovirus (PCV-2) from pigs withpostweaning multisystemic wasting syndrome in different geographicregions of North America and development of a differentialPCR-restriction fragment length polymorphism assay to detect anddifferentiate between infections with PCV-1 and PCV-2,” J. Clin.Microbiol. 38: 2494-503 (2000)). PCR products from selected animals ineach group were sequenced to verify the origin of the virus infectingpigs.

Example 20 Immunohistochemistry (IHC)

IHC detection of PCV2-specific antigen was performed on lymph nodetissues collected from all pigs necropsied at 21 and 49 DPIs. A rabbitpolyclonal antiserum against PCV2 was used for the IHC, according to thegeneral procedures described previously (S. D. Sorden et al.,“Development of a polyclonal-antibody-based immunohistochemical methodfor the detection of type 2 porcine circovirus in formalin-fixed,paraffin-embedded tissue,” J. Vet. Diagn. Invest. 11:528-530 (1999)).

Based on the IHC staining of PCV2-specific antigen, lymphoid tissuesfrom the uninoculated control, PCV1 and PCV2-1 inoculated pigs werenegative for PCV2 antigen. PCV2 antigen was detected in lymphoid tissuesof 7 out of 8 animals in the PCV2 inoculated group. PCV2 antigen wasalso detected in lymphoid tissue of 1 out of 7 pigs from the chimericPCV1-2 inoculated group.

Examples 21-24

The following general materials and methods are employed in Examples21-24:

(1) Virus and Cell.

The PCV1 virus used in this invention was originally isolated from aPK-15 cell line (ATCC CCL-33) (M. Fenaux et al., 2002, supra). The PCV2virus used herein was originally isolated from a spleen tissue sample ofa pig with naturally occurring PMWS (isolate 40895) (M. Fenaux et al.,2002, supra; M. Fenaux et al., 2000, supra,). The PK-15 cell line usedherein was free of PCV1 contamination (M. Fenaux et al., 2002, supra).

(2) Serial Passages of PCV2 In Vitro.

A homogenous PCV2 virus stock, designated passage 1 (VP1), was generatedby transfection of PK-15 cells with the PCV2 infectious DNA clone aspreviously described (M. Fenaux et al., 2002, supra). The VP1 PCV2 virusstock was then serially passaged for 120 times in PK-15 cells. Theinfected cells, when reaching confluency, were subcultured at anapproximate 1 to 3 ratio in minimum essential medium (MEM) with Earle'ssalts and L-glutamine supplemented with 2% fetal calf serum (FCS) and 1×antibiotic (Invitrogen, Inc., CA). For every 10 to 15 passages, theinfected cells were harvested by frozen and thawed three times, and usedto inoculate a new PK-15 culture. The newly infected culture was thenpassed 10 to 15 times by subculturing before repeating the freeze-thawprocedure. This procedure was repeated until it reached passage 120(VP120). The virus harvested at each passage was stored at −80 forfurther analyses.

(3) Biological Characterization of PCV1, PCV2 VP1, and PCV2 VP120Viruses in PK-15 Cells.

A one-step growth curve was performed to determine the comparativegrowth ability of PCV1, PCV2 VP1, and PCV2 VP120 in vitro. PK-15 cellswere grown on six 12-well plates. The plates were infected, induplicate, with PCV1, PCV2 VP1 or PCV2 VP120 at a multiplicity ofinfection (M.O.I.) of 0.1, respectively. After 1 hour absorption, theinoculum was removed and the cell monolayer was washed five times withphosphate buffered saline (PBS). Maintenance MEM media (2% bovine calfserum and 1× antibiotics) was subsequently added to each well, and theinfected cell cultures were continuously incubated at 37° C. with 5%CO₂. Every 12 hours, the media and infected cells from duplicate wellsof each inoculated group were harvested and stored at −80° C. untilvirus titration. The infectious titers of PCV1 and PCV2 virusescollected at different time points were determined by immunofluorescentassays (IFA) specific for PCV1 or PCV2 as previously described (M.Fenaux et al., 2002, supra; M. Fenaux et al., 2003, supra).

(4) Genetic Characterization of PCV2 Viruses at Different Passages.

PCV2 viruses harvested from passages 1, 30, 60, 90, and 120 weregenetically characterized by determining the complete genomic sequencesof the viruses from each passage. Viral DNA was extracted from 100 μl ofthe cell culture materials collected at passages 1, 30, 60, 90, and 120by using DNAzol reagent according to the manufacturer's protocol(Molecular Research Center, Cincinnati, Ohio). The extracted DNA wasresuspended in DNase-, RNase- and proteinase-free water. To amplify theentire genome, three pairs of PCV2 specific primers were used to amplifythree overlapping fragments: primer pair PCV2.2B(5′-TCCGAAGACGAGCGCA-3′, set forth in SEQ ID NO:27) and PCV2.2A(5′-GAAGTAATCCTCCGATAGAGAGC-3′, set forth in SEQ ID NO:28), primer pairPCV2.3B (5′-GTTACAAAGTTATCATCTAGAATAACAGC-3′, set forth in SEQ ID NO:29)and PCV2.3A (5′-ATTAGCGAACCCCTGGAG-3′, set forth in SEQ ID NO:30), andprimer pair PCV2.4B (5′-AGAGACTAAAGGTGGAACTGTACC-3′, set forth in SEQ IDNO:31) and PCV2.4A (5′-AGGGGGGACCAACAAAAT-3′, set forth in SEQ IDNO:32). The PCR reaction consisted of 38 cycles of denaturation at 94°C. for 1 min, annealing at 46° C. for 30 sec, and extension at 72° C.for 2 min, followed by a final extension at 72° C. for 7 min. The PCRproducts of expected size were excised from 0.8% agarose gels followedby purification with a Geneclean Kit (Bio 101, Inc., La Jolla, Calif.).The PCR products were directly sequenced for both strands using the PCRprimers. The nucleotide and amino acid sequences were compiled andanalyzed with the publicly accessible MacVector program (OxfordMolecular Ltd., Beaverton, Oreg.) using Clustal alignment. The completesequence of PCV2 VP120 was compared to PCV2 VP1 and 91 other PCV2isolates as well as 4 PCV1 isolates available in the GenBank database.

(5) Experimental Characterization of the Serially-Passaged PCV2 VP120,and VP1.

To determine the pathogenic potential of the VP120 PCV2, thirty-one SPFpigs of 3 to 4 weeks of age were randomly assigned to 3 groups, andhoused separately. Prior to the inoculation, serum samples from allpiglets were tested by PCR for the presence of PCV1 or PCV2 DNA. Tomaximize the efficiency of inoculation, each pig was inoculated with 1ml of the inoculum intramuscularly and 3 ml intranasally. The ten pigsin Group 1 were each inoculated with PBS buffer as negative controls.Eleven pigs in Group 2 each received 10^(4.9) TCID₅₀ of PCV2 VP120, andten pigs in Group 3 each received 10^(4.9) TCID₅₀ of PCV2 VP1. All pigswere monitored for clinical signs of disease. Serum samples werecollected from each pig at −1, 7, 14, 21, 28, 35 and 42 days postinoculation (DPI). At 21 DPI, 5 randomly selected pigs from each groupwere necropsied. The remaining pigs in each group were necropsied at 42DPI.

(6) Clinical Evaluation.

Pigs were weighed at −1, 7, 14, 21, 28, 35 and 42 DPI. Rectaltemperatures and clinical scores, ranging from 0 to 6 (0=normal;6=severe) (M. Fenaux et al., 2002, supra), were recorded every other dayfrom 0 to 42 DPI. Clinical observations, including evidence of centralnervous system disease, liver disease (icterus), musculoskeletaldisease, and changes in body condition, were recorded at two-dayintervals. All clinical evaluations were performed by a team of twopeople to confirm observations.

(7) Gross Pathology and Histiopathology.

Complete necropsies were performed on all pigs. The necropsy team wasblinded to the infection status of the pigs. The percentage of lung withgrossly visible pneumonia was estimated for each pig based on apreviously described scoring system (M. Fenaux et al., 2002, supra).Lesions such as the enlargement of the lymph nodes (ranging from 0 fornormal to 3 for three times normal size) were scored separately.Sections for histopathologic examination were taken from the nasalturbinate, lungs (five sections, see M. Fenaux et al., 2002, supra),heart, brain, lymph nodes (tracheobronchial, iliac, mesenteric,subiliac, and superficial inguinal), tonsil, liver, thymus, spleen,pancreas, and kidney. The tissues were examined in a blinded fashion andgiven a subjective score for severity of lung, lymph node, and liverlesions (M. Fenaux et al., 2002, supra). Lung scores ranged from 0(normal) to 3 (severe lymphohistiocytic interstitial pneumonia). Liverscores ranged from 0 (normal) to 3 (severe lymphohistiocyticinterstitial hepatitis). Lymph node scores were an estimated amount oflymphoid depletion of follicles ranging from 0 (normal or no lymphoiddepletion) to 3 (severe lymphoid depletion and histiocytic replacementof follicles) (M. Fenaux et al., 2002, supra).

(8) Serology.

Blood samples were collected from all pigs at −1, 7, 14, 21, 28, 35, and42 DPI. Serum antibodies to PCV2 were detected by a modified indirectELISA based on the recombinant ORF2 capsid protein of PCV2 (P.Nawagitgul et al., 2002, supra). Serum samples with a sample/positive(S/P) ratio above 0.2 were considered seropositive for PCV2.

(9) Quantitative Real-Time PCR.

Quantitative real-time PCR was performed to determine PCV2 virus loadsin serum samples collected at −1, 7, 14, 21, 28, 35, and 42 DPI and inlymphoid tissue samples collected at 21 DPI and 42 DPI. Primer pair MCV1(5′-GCTGAACTTTTGAAAGTGAGCGGG-3′, set forth in SEQ ID NO:17) and MCV2(5′-TCACACAGTCTCAGTAGATCATCCCA-3′, set forth in SEQ ID NO:18) (M. Fenauxet al., 2000, supra) was used for the quantitative real-time PCR. ThePCR reaction was performed in the presence of intercalating SYBR greendye (Molecular Probes, Inc. Eugene, Oreg.) as described herein. Astandard dilution series with a known amount of pBluescript plasmidcontaining a single copy of the PCV2 genome (M. Fenaux et al., 2002,supra) was run simultaneously in each real-time PCR reaction to quantifythe virus genomic copy numbers.

(10) Immunohistochemistry (IHC).

IHC detection of PCV2 specific antigen was performed on lymph node,spleen, tonsil and thymus tissues collected during necropsy at 21 and 42DPI as described hereinabove (M. Fenaux et al., 2002, supra). The amountof PCV2 antigen distributed in the lymphoid tissues was scored in ablinded fashion by assigning a score of 0, if no signal, to 3 for astrong positive signal (S. D. Sorden et al., “Development of apolyclonal-antibody-based immunohistochemical method for the detectionof type 2 porcine circovirus in formalin-fixed, paraffin-embeddedtissue,” J. Vet. Diagn. Invest. 11:528-530 (1999)).

(11) Statistical Analysis.

All statistical analyses were performed using the SAS®-system (Version8.02, SAS institute Inc. Cary N.C. 27513). Growth characteristics ofviruses were compared by regressive analyses using the GLM procedure.Serum samples S/P ratios were compared by analysis of variance, with theMIXED procedure. The model included effects of inoculum, DPI, and theirinteraction. S/P ratios were dichotomized to presence/absence ofseroconversion at S/P=0.20 and analyzed by logistic regression using themethod of generalized equations in the SENROD procedure. Mean viralgenomic copy numbers in serum and lymph nodes of piglets in Groups 2 and3 were compared by the Kruskal-Wallis test using the NPAR1WAY procedureand/or by analysis of variance of ranked data, followed by a Bonferronitest of multiple mean ranks, using the GLM procedure. Serology andviremia data were analyzed for all pigs up to 21 DPI, and separately forthose pigs necropsied at 42 DPI. Clinical sign scores were dichotomizedto presence/absence of clinical signs for each examination date, and perpig over the entire period of study and compared between groups byFisher's Exact test using the FREQ procedure, and by logistic regressionusing LOGISTIC procedure. Gross pathologic and histopathologic scoreswere compared by the Kruskal-Wallis test using the NPAR1WAY procedureand/or by analysis of variance using the GLM procedure, followed by aBonferroni test of multiple means. Proportion of pigs with gross andhistopathologic lesions in various tissues was compared between groupsby Fisher's Exact Test using the FREQ procedure.

Example 21 Comparison of the Replication of PCV2 VP120 and PCV2 VP1 inPK-15 Cells

To determine the growth characteristics of PCV1, PCV2 VP1 and VP120,one-step growth curves were performed in duplicate simultaneously forPCV1, PCV2 VP1 and PCV2 VP120. The infectious titers of virusescollected at 12 hour intervals were determined by IFA (FIG. 12). Theinitial titers after infection at 12 h postinoculation were about10^(1.5) TCID₅₀/ml for all three viruses. The infectious titers of PCV1and PCV2 VP120 compared to PCV2 VP1 increased differently (p=0.0053)from 12 to 96 h. By 96 h postinfection, PCV1 and PCV2 VP120 had titersof 10^(3.66) and 10^(3.75) TCID₅₀/ml, whereas the PCV2 VP1 was 10^(2.83)TCID₅₀/ml (FIG. 12). It was demonstrated that PCV2 VP120 surprisinglyreplicated more efficiently in PK-15 cells than PCV2 VP1.

Example 22 Identification of Two Amino Acid Mutations within the PCV2Capsid Protein During Serial Passages

The complete genomes of PCV2 passage numbers 1, 30, 60, 90, and 120 wereamplified and sequenced. Sequence analyses revealed that there were atotal of 2 nucleotide and 2 amino acid mutations in the entire genomeafter 120 passages. The first mutation appeared in passage 30 (VP30) inwhich a proline was substituted for an alanine at position 110 of thecapsid (P110A) (FIG. 13). This mutation was also present during theremaining passages. A second mutation from arginine to serine atposition 191 of the capsid (R191S) was identified at passage 120 but notin lower passages (FIG. 13). The amino acid mutations were the result ofthe corresponding mutations that occurred in the genome in nucleotidepositions 328 (C to G) and 573 (A to C). In position 328 of thenucleotide sequence, cytosine changes to guanine (C to G) leading to theamino acid change of P110A. In position 573, adenine changes to cytosine(A to C) leading to the second amino acid change of R191S.

By comparing all known PCV1 and PCV2 sequences in the Genbank including91 PCV2 and 4 PCV1 isolates, it was found that that the P110A mutationis unique (FIG. 13) as all known PCV1 and PCV2 isolates have a prolineat residue 110 of the capsid protein. The serine in the R191S mutationis also unique. However, the amino acid in the 191 position is oftenvariable: PCV2 isolates of North American origin have an arginine, PCV2isolates of Canadian and French origins have glycine, and PCV2 isolatesof Spanish, Taiwanese and German origins have an alanine. Allnonpathogenic PCV1 isolates have a threonine residue (FIG. 13).

Example 23 Comparison of the Viremia Length and Virus Loads of PCV2VP120 Virus and PCV2 VP1 Virus in Sera of Infected Pigs

Serum samples were collected from all control and inoculated pigs at −1,7, 14, 21, 28, 35, and 42 DPI and assayed for PCV2 viremia byquantitative real-time PCR and for anti-PCV2 antibody by ELISA. Prior toinoculation at −1 DPI, serum samples from all pigs were tested negativefor PCV2 DNA.

The Group 1 negative control pigs were negative for PCV2 viremiathroughout the study (see Table 11, below). All pigs in Group 1 haddetectable PCV2 maternal antibodies at −1 DPI, which all waned by 21DPI. Seroconversion to PCV2 was not detected in any of the ten negativecontrol pigs (see Table 12, below).

In the PCV2 VP120 inoculated Group 2 pigs, viremia was first detected inone of eleven pigs at 7 DPI (Table 11, FIG. 14). A total of 4 pigs inGroup 2 were viremic during the study. The average length of continuousviremia was 1.6 weeks. By 35 DPI, all Group 2 pigs seroconverted to PCV2(Table 12).

In the PCV2 VP1 inoculated Group 3 pigs, viremia was first detected inseven of ten pigs at 7 DPI (Table 11, FIG. 14). Nine out of the ten pigsin Group 3 became viremic for PCV2 during the study and the averagelength of continuous viremia was 3 weeks. All animals in Group 3seroconverted to PCV2 by 35 DPI (Table 12).

The range of PCV2 genomic copy numbers per ml of serum in positivesamples was 8,840 to 274,800 in PCV2 VP120 inoculated Group 2 pigs, and26,520 to 120,000,000 in PCV2 VP1 inoculated Group 3 pigs (FIG. 14).PCV2 genomic copy loads per ml of serum were greater in Group 3 thanthat in Group 2 pigs up to 21 DPI (p=0.0003) and 42 DPI (p=0.039).However, PCV2 DNA was recovered from lymph nodes of only 3/11 Group 2and 2/10 Group 3 pigs, and the median PCV2 genomic copy loads per mg oftracheobronchial lymph node (TBLN) did not differ between Groups 2 and 3(p=0.72). The virus recovered from the sera and TBLN of 4 selected pigsin Groups 2 and 3 were sequenced, and sequence analyses revealed thatthe recovered viruses originated from the inocula.

The S/P ratios of PCV2 antibodies differed between Groups 1, 2 and 3(p<0.0001) and over time (p<0.0001). It was shown that the PCV2 VP120virus significantly reduced viremia length and virus loads in sera ofinfected pigs compared to the PCV2 VP1 virus.

TABLE 11 Detection of Viremia by Real-Time PCR in Sera of Inoculated andControl Pigs No. of pigs positive/no. tested^(a) Days post inoculationGrp. Inocula −1 7 14 21 28 35 42 total 1 Control 0/10 0/10 0/10 0/10 0/50/5 0/5 0/10 2 PCV2 VP120 0/11 1/11 2/11 3/11 0/6 1/6 1/6 4/11 3 PCV2 VP1 0/10 7/10 8/10 8/10 3/5 3/5 1/5 9/10 ^(a)Five pigs per group werenecropsied at 21 day post inoculation (DPI) and the remaining pigs werenecropsied at 42 DPI.

TABLE 12 Seroconversion to PCV2 Antibodies in Pigs Inoculated with PCV2Passages 1 (VP1) and 120 (VP120) No. of pigs positive/no. tested^(a)Days post inoculation Grp. Inoculum −1 7 14 21 28 35 42 1 Control 10/10^(b) 1/10 1/10 0/10 0/5 0/5 0/5 2 PCV2 VP120  3/15 ^(b) 0/11 0/11 0/110/6 6/6 6/6 3 PCV2 VP 1  1/10 ^(b) 0/10 1/10 2/10 4/5 5/5 5/5 ^(a)Fivepigs per group were necropsied at 21 day post inoculation (DPI) and theremaining pigs were necropsied at 42 DPI. ^(b) Maternal antibodies weredetectable at −1 DPI but waned in all groups between 7 and 21 DPI.

Example 24 Attenuation of PCV2 VP120 Virus in Pigs

Mild clinical signs (sneezing and rough coat) were noted in some animalsfrom all three groups. Two of 10 non-inoculated, and all of 21inoculated pigs developed clinical signs (p=0.051). Up to 21 DPI, Groups2 and 3 pigs were 58 (95% C.I.: [13.1; 255.0]) and 41 [9.3;178.0] timesmore likely to show mild clinical signs at any examination date thannegative control pigs, with no difference between pigs of Groups 2 and 3(OR_(3vs2): 1.4[0.8;2.6]). For the 16 pigs that were necropsied at 42DPI, when evaluated over the entire study period, Groups 2 and 3 pigsagain were more likely to show mild clinical signs than negative controlpigs (OR_(2vs1): 20.4[4.6;90.1]; OR_(3vs1): 71.6 [16.0;320.8]) withGroup 3 pigs being 3.5 [1.9;6.6] times more likely to show mild clinicalsigns than Group 2 pigs. There were no differences in weight gain(p=0.081) or mean rectal temperatures (p>0.05) among any of the groups.

At necropsies, lymph nodes of 2/5 pigs in Group 1 were mildly enlarged,however this was not associated with PCV2 infection as evidenced by thelack of PCV2 DNA or seroconversion. At 42 DPI necropsy, all Group 1 pigshad normal lymph nodes (see Table 13, below). Group 2 pigs inoculatedwith PCV2 VP120 had mild to moderately enlarged lymph nodes at both 21and 42 DPI (Table 13). The lymph nodes in Group 3 pigs were moderatelyto severely enlarged at both 21 and 42 DPI (Table 13). Pigs inoculatedwith PCV2 VP1 had visible gross pneumonia at 21 DPI. Visible grosspneumonia was not found in Group 1 or 2 pigs at either 21 or 42 DPI.

Microscopic lung lesions characterized by mild peribronchiolarlymphoplasmacytic and histiocytic bronchointerstitial pneumonia andliver lesions characterized by mild lymphoplasmacytic hepatitis weredetected in pigs of all groups (see Table 14, below). Mild lymphoiddepletion (LD) of lymph node follicles was detected in 0/5 Group 1 pigsat 21 and 42 DPI, in 3/5 (21 DPI) and in 2/6 (42 DPI) Group 2 pigs, in4/5 (21 DPI) and 5/5 (42 DPI) Group 3 pigs (Table 14). Mild histiocyticreplacement (HR) of lymph node follicles was not observed in Group 1pigs. In Group 2, mild HR was observed in the lymph nodes of 0/5 pigs at21 DPI and 2/6 pigs at 42 DPI. In Group 3, mild to moderate HR of thelymph nodes was observed in 3/5 pigs at both 21 and 42 DPI. The tonsiland spleen tissue follicles of the Group 1 pigs were free of LD and HRat 21 or 42 DPI. Mild LD of the tonsil follicles was found in 2/5 pigsin Group 2 at 21 DPI. Mild to moderate LD of the tonsil follicles wasfound in 2/5 pigs in Group 3 at 21 DPI and mild HR of the tonsil tissuein 1/5 pigs in Group 3 at both 21 and 42 DPI. Mild LD of the spleenfollicles was observed in 2/5 and 1/6 pigs in Group 2 at 21 and 42 DPI,respectively. Mild to moderate LD of the spleen follicles was noted in4/5 in Group 3 pigs at both 21 and 42 DPI. In Group 2 pigs, mild HR ofthe spleen tissue follicles was found in 1/6 pigs at 42 DPI. In Group 3,4/5 pigs at 21 DPI and 3/5 pigs at 42 DPI had mild to moderate HR of thespleen tissue follicles (Table 14). Presence of lesions in other tissuesand organs are summarized in Table 14.

At necropsies (21 and 42 DPI), PCV2 antigen was not detected by IHC inthe lymphoid tissues of the negative control Group 1 pigs. In the PCV2VP120 inoculated Group 2, low amounts of PCV2 antigen was detected inspleen tissues of 1/5 pigs at 21 DPI, in lymph node tissues of 2/6 pigsat 42 DPI, and in tonsil tissues of 3/6 pigs at 42 DPI (see Table 15,below). In the PCV2 VP0 inoculated Group 3, low-to-high amounts of PCV2antigen were detected in lymph node tissues of 5/5 pigs, in tonsiltissues of 4/5 pigs, and in spleen tissues of 4/5 pigs at 21 DPI. At 42DPI, low-to-moderate amounts of PCV2 antigen were detected in PCV2 VP0inoculated Group 3 (Table 15).

All gross pathologic and histopathologic scores at 21 DPI and 42 DPIwere compared by analysis of variance using the GLM procedure followedby a Bonferroni test of multiple means. At 21 DPI, Groups 1 and 2 meanscores are similar (p=1.00) but differ from the mean scores of Group 3(p=0.0032). By 42 DPI, the mean scores of Group 1 differ from Group 2(p=0.0083) and Group 3 (p=0.0001), and the Group 2 mean scores aremilder than those of Group 3 (p=0.0274) (Tables 13-15).

TABLE 13 Gross Lymph Node and Lung Lesions in Control and InoculatedPigs No. of pigs with enlarged No. of pigs with gross lymph nodes^(a)pneumonia lesions Group Inoculum 21 DPI 42 DPI 21 DPI 42 DPI 1 Control2/5(0.4^(b))^(Ic) 0/5(0.0)^(I) 0/5(0.0)^(I) 0/5(0.0) 2 PCV2 VP1202/5(0.4)^(I) 4/6(1.3)^(II) 0/6(0.0)^(I) 0/5(0.0) 3 PCV2 VPI5/5(2.2)^(II) 5/5(2.6)^(II) 2/5(2.6)^(II) 0/5(0.0) ^(a)Five pigs fromeach group was necropsied at 21 days post inoculation (DPI) and theremaining pigs were necropsied at 42 DPI. ^(b)Values in parentheses arethe mean scores of estimated lymph node enlargement (0 = normal to 3severely enlarged and discolored) and mean percentage of lungs affectedby gross visible pneumonia (0-100%) ^(c)Different superscripts (I, II)indicate different mean value score between groups (p < 0.05).

TABLE 14 Distribution of Histopathological Lesions in Different Tissuesand Organs from Control and Inoculated Pigs No. of pigs positive/no. ofpigs tested Lymph nodes Tonsil Spleen Group Inocula DPI^(a) Lung LD^(¶c)HR LD^(†) HR LD^(¶) HR^(†) Liver Kidney Heart 1 Control 21 0/5(0.0)^(b)0/5(0.0) 0/5(0.0) 0/5(0.0) 0/5(0.0) 0/5(0.0) 0/5(0.0) 0/5(0.0) 1/5 1/542 3/5(0.6) 0/5(0.0) 0/5(0.0) 0/5(0.0) 0/5(0.0) 0/5(0.0) 0/5(0.0)1/5(0.2) 1/5 0/5 2 PCV2 21 2/5(0.4) 3/5(0.6) 0/5(0.0) 2/5(0.4) 0/5(0.0)2/5(0.4) 0/5(0.0) 3/5(0.6) 2/5 0/5 VP120 42 5/6(1.0) 2/6(0.3) 2/6(0.3)0/6(0.0) 0/6(0.0) 1/6(0.2) 1/6(0.2) 0/6(0.0) 2/6 0.5 3 PCV2 21 2/5(0.4)4/5(1.2) 3/5(1.0) 5/5(1.2) 1/5(0.2) 4/5(1.0) 4/5(1.0) 3/5(1.2) 3/5 3/5VP0 42 5/5(1.4) 5/5(1.4) 3/5(0.8) 0/5(0.0) 1/5(0.2) 4/5(1.0) 3/5(0.6)3/6(0.6) 1/5 2/5 ^(a)DPI, Days postinoculation ^(b)Values in parenthesesare mean histological scores for interstitial pneumonia and forinterstitial hepatitis and lymphoid depletion (LD) and histiocyticreplacement (HR) for lymph nodes, tonsils and spleen. ^(c)Indicatesdifference (p < 0.05) using Fisher's Exact test between Groups 1, 2, and3 in severity of respective histopathological lesion with symbol ^(†)at21 DPI and symbol ^(¶)at 42 DPI necropsies.

TABLE 15 Immunohistiochemical Detection of PCV2 Antigen in Lymph Nodes,Tonsils and Spleen of Inoculated and Control Pigs No. of pigspositive/no. tested^(b) Group Inocula DPI^(a) Lymph node^(†b) Tonsil^(†)Spleen 1 Control 21 0/5 (0.0)^(c) 0/5 (0.0) 0/5 (0.0) 42 0/5 (0.0) 0/5(0.0) 0/5 (0.0) 2 PCV2 VP120 21 0/5 (0.0) 0/5 (0.0) 1/5 (0.2) 42 2/6(0.3) 3/6 (0.5) 0/6 (0.0) 3 PCV2 VPI 21 5/5 (1.6) 4/5 (1.0) 4/5 (1.2) 423/5 (0.8) 2/5 (0.4) 2/5 (0.4) ^(a)DP1, days postinoculation^(b)Indicates difference (p < 0.05) using Fisher's Exact test betweenGroups 1, 2, and 3 of PCV2 antigen presence in respective tissues withsymbol † at 21 DPI. ^(c)Value in parentheses are the mean scores of theamounts of PCV2 antigen in lymphoid tissues (ranging from 0, no antigendetected, to 3, high levels of antigen).

In the foregoing, there has been provided a detailed description ofparticular embodiments of the present invention for the purpose ofillustration and not limitation. It is to be understood that all othermodifications, ramifications and equivalents obvious to those havingskill in the art based on this disclosure are intended to be includedwithin the scope of the invention as claimed.

The invention claimed is:
 1. A recombinant porcine circovirus type 1encoding the ORF2 capsid protein of a porcine circovirus type 2(PCV1-2).
 2. The PCV1-2 virus according to claim 1, wherein the PCV1-2virus is made from a nucleotide sequence having at least 95% homology tothe nucleotide sequence of SEQ ID NO:
 2. 3. The PCV1-2 virus accordingto claim 2, wherein the PCV1-2 virus is made from a nucleotide sequenceset forth in SEQ ID NO: 2 or its complementary strand.
 4. The PCV1-2virus according to claim 1, wherein the PCV1-2 virus is inactivated. 5.An inactivated vaccine for use in immunizing pigs against PCV2 infectionor postweaning multisystemic wasting syndrome (PMWS) caused by PCV2,wherein the vaccine comprises the inactivated PCV1-2 virus according toclaim
 4. 6. The vaccine according to claim 5, further comprising anadjuvant.
 7. The vaccine according to claim 5, further comprising asuspending agent or an emulsifying agent.
 8. The vaccine according toclaim 5, further comprising at least one additional porcine antigen. 9.The vaccine according to claim 8, wherein the additional porcine antigenis an infectious swine agent.
 10. The vaccine according to claim 5,wherein the inactivated PCV1-2 virus is suspended in a physiologicallyacceptable carrier or solvent system.
 11. The vaccine according to claim5, further comprising a preservative.
 12. A method of immunizing a pigagainst PCV2 infection or postweaning multisystemic wasting syndrome(PMWS) caused by PCV2, comprising administering to the pig animmunologically effective amount of the vaccine according to claim 5.13. The method according to claim 12, which comprises administering thevaccine in a single dose or in repeated doses.
 14. The method accordingto claim 13, which comprises administering the vaccine in a single dose.15. The method according to claim 12, which comprises administering thevaccine intranasally, transdermally, or parenterally.
 16. The methodaccording to claim 15, wherein the parenterally administered vaccine isadministered intramuscularly, intravenously, intraperitoneally, orintradermally.
 17. The method according to claim 16, which comprisesadministering the vaccine intramuscularly.
 18. A method of preparing avaccine according to claim 5, comprising the steps of: (a) replacing theORF2 gene in the genome of PCV1 with the ORF2 gene of PCV2 to form aninfectious chimeric nucleic acid molecule of PCV1-2; (b) infecting cellswith the infectious chimeric nucleic acid molecule of step (a) toproduce live recombinant PCV1-2 virus expressing the ORF2 capsid proteinof PCV2; and (c) inactivating the live recombinant PCV1-2 virus of step(b).
 19. The method according to claim 18, wherein step (c) comprisesinactivating the live recombinant PCV1-2 virus with an inactivatingagent.
 20. The method according to claim 19, further comprising step (d)adding a suspending agent to form a suspension of the inactivated PCV1-2virus and step (e) combining the suspension with an adjuvant, at leastone additional porcine antigen or both an adjuvant and at least oneadditional porcine antigen.
 21. The method according to claim 20,wherein the additional porcine antigen is an infectious swine agent.